IGF-binding protein-derived peptide or small molecule

ABSTRACT

New compositions based on IGF-binding protein sequences are provided. New tools for high-throughput research are provided. New methods for the treatment of human disease are provided. IGFBP-3-derived peptide or small molecule is administered to subjects having disease, thereby alleviating the symptoms of the disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/383,999, filed Mar. 7, 2003, which is a continuation-in-part of U.S.patent application Ser. No. 10/264,672, filed Oct. 4, 2002, which is acontinuation-in-part of U.S. patent application Ser. No. 10/215,759,filed Aug. 9, 2002, which claims priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application Ser. No. 60/323,267, filed Sep. 18,2001, all of which are incorporated by reference in their entirety.

TECHNICAL FIELD

The invention relates to the compositions and methods of use of peptidesor small molecules in the treatment of disease, as well as inhigh-throughput screening and other discovery and research applications,particularly to the use of metal-binding peptides derived from sequencespresent in the CD74-homology domain of insulin-like growth factorbinding protein-3 (IGFBP-3).

BACKGROUND ART

Growth factors are polypeptides which stimulate a wide variety ofbiological responses (e.g. DNA synthesis, cell division, expression ofspecific genes, etc.) in a defined population of target cells. A varietyof growth factors have been identified, including the transforminggrowth factor beta family (TGF-βs), epidermal growth factor andtransforming growth factor alpha (the TGF-αs), the platelet-derivedgrowth factors (PDGFs), the fibroblast growth factor family (FGFs) andthe insulin-like growth factor family (IGFs), which includes IGF-I andIGF-II. Many growth factors have been implicated in the pathogenesis ofcancer.

IGF-I and IGF-II (the “IGFs”) are related in amino acid sequence andstructure, with each polypeptide having a molecular weight ofapproximately 7.5 kilodaltons (kDa). IGF-I mediates the major effects ofgrowth hormone, and is thus the primary mediator of growth after birth.IGF-I has also been implicated in the actions of various other growthfactors, since the treatment of cells with such growth factors leads toincreased production of IGF-I. In contrast, IGF-II is believed to have amajor role in fetal growth. Both IGF-I and IGF-II have insulin-likeactivities (hence their names), and are mitogenic (stimulate celldivision).

IGF-I has been found to stimulate the growth of cells from a number ofdifferent types of cancer (Butler et al., 1998 Cancer Res.58(14):3021-3027; Favoni R E, et al., 1998, Br. J. Cancer 77(12):2138-2147). IGF-I has additionally been found to exert anti-apoptoticeffects on a number of different cell types, including tumor cells(Giuliano M, et al., 1998 Invest Ophthalmol. Vis. Sci. 39(8): 1300-1311;Zawada W M, et al., 1998, Brain Res. 786(1-2): 96-103; Kelley K W, etal., 1998, Ann. N.Y. Acad. Sci. 840: 518-524; Toms S A, et al., 1998, J.Neurosurg. 88(5): 884-889; Xu F, et al., 1997, Br. J. Haematol. 97(2):429-440). Prospective studies have implicated IGF-I as a risk factor forcancers of the prostate, breast, and colon, while IGFBP-3, the majorcirculatory binding protein for IGFs, appears to have a protectiveeffect (10-12, 28, 29). A variety of other observations further supportthe idea that the relative balance of IGFBP-3 to other IGF-bindingproteins (notably IGFBP-2) is somehow instrumental in the control oftumor cell growth, both in vitro and in vivo (7-9). Recent evidence alsosuggests that IGFBP-3 may play a central role in the growth (13-17) andapoptosis (14) of tumor cells in an IGF-independent manner.

Approximately half of the 1.3 million patients diagnosed with cancereach year in the U.S. have (or will be at risk for) systemic disease.Chemotherapy is the most common therapeutic approach for these patients(34). Most chemotherapeutic agents are effective primarily againstdividing cells, and myelosuppression is often the dose-limitingtoxicity. Chemical agents fall into several categories and havedifferent mechanisms of action but, at effective doses, most haveside-effects which seriously impact the patient's quality of lifedoxorubicin (ADRIAMYCIN®), irinotecan (CPT-11), paclitaxel (TAXOL®),cisplatin, tamoxifen, methotrexate and 5-fluorouracil are popular agentsused to treat a variety of cancers, sometimes in combination. Inaddition to myelosuppression, gastrointestinal effects, mucositis,alopecia, and (in the case of doxorubicin) cardiac toxicities are alsoobserved with these agents (34). Clearly, it would be of interest tofind ways to make tumor cells selectively sensitive to these chemicalagents.

Almost all IGF circulates in a non-covalently associated complex ofIGF-I, insulin-like growth factor binding protein 3 (IGFBP-3) and alarger protein subunit termed the acid labile subunit (ALS), such thatvery little free IGF-I is detectable. The ternary complex is composed ofequimolar amounts of each of the three components. ALS has no directIGF-binding activity and appears to bind only to the IGF/IGFBP-3 complex(Baxter et al., J. Biol. Chem. 264(20):11843-11848, 1989), although somereports suggest that IGFBP-3 can bind to rat ALS in the absence of IGF(Lee et al., Endocrinology 136:4982-4989, 1995). The ternary complex ofIGF/IGFBP-3/ALS has a molecular weight of approximately 150 kDa and hasa substantially increased half-life in circulation when compared tobinary IGF/IGFBP-3 complex or IGF alone (Adams et al., Prog. GrowthFactor Res. 6(2-4):347-356; presented October 1995, published 1996).This ternary complex is thought to act “as a reservoir and a buffer forIGF-I and IGF-II preventing rapid changes in the concentration of freeIGF” (Blum et al. (1991), “Plasma IGFBP-3 Levels as Clinical Indicators”in MODERN CONCEPTS OF INSULIN-LIKE GROWTH FACTORS, pp. 381-393, E. M.Spencer, ed., Elsevier, N.Y.). While there is essentially no excess(unbound) IGFBP-3 in circulation, a substantial excess of free ALS doesexist (Baxter, J. Clin. Endocrinol. Metab. 67:265-272, 1988).

How IGFBP-3 mediates its cellular effects is not well understood,although there is indirect evidence to suggest that it mediates some ofthe effects of p53, a well-characterized tumor suppressor (Ferry et al.,(1999) Horm Metab Res 31(2-3):192-202). IGFBP-3 is mobilized to thenucleus of rapidly growing cells (Schedlich, et al., (1998) J. Biol.Chem. 273(29):18347-52; Jaques, et al., (1997) Endocrinology138(4):1767-70). A useful step toward defining the functionalinteractions of IGFBP-3 would be to identify protein domains involved inthe ability of IGFBP-3 to specifically bind a surprisingly large arrayof intracellular and extracellular targets. Known targets include:IGF-I, IGF-II, insulin (under some conditions), acid-labile subunit(ALS), plasminogen, fibrinogen, transferrin, lactoferrin, collagen TypeIa, prekallikrein, RXR-alpha, viral oncoproteins, heparin, specificproteases, cellular receptors, a number of intracellular targetsidentified in two-hybrid screens, and components of the nuclearlocalization transport machinery (Mohseni-Zadeh and Binoux (1997)Endocrinology 138(12):5645-8; Collett-Solberg, et al. (1998) J. Clin.Endocrinol Metab. 83(8):2843-8; Rajah, et al. (1995) Prog. Growth FactorRes. 6(2-4):273-84; Fowlkes and Serra (1996) J. Biol. Chem.271:14676-14679; Campbell, et al. (1999) J. Biol Chem. 274(42):30215-21;Durham, et al. (1999) Horm Metab Res 31(2-3):216-25; Campbell, et al.(1998) Am J Physiol. 275(2Pt 1):E321-31).

IGFBP-3 has three major domains, roughly corresponding to exons 1, 2 and3+4 of the IGFBP-3 gene, respectively. The C-terminal domain of IGFBP-3(Domain 3), which contains sequences homologous to a motif found in CD74(invariant chain) and a number of other proteins, appears to be involvedin IGFBP-3's ability to interact with serum, extracellular matrix, andcell surface components. Peptides made to sequences in this region havepreviously been shown to interfere with the binding of IGFBP-3 to anumber of its known ligands, including RXR-alpha, transferrin, ALS,plasminogen, fibrinogen and pre-kallikrein (Liu, et al, J. Biol. Chem.275: 33607-13, 2000; Weinzimer, et al, J. Clin. Endocrinol. Metab. 86:1806-13, 2001; Campbell, et al, Am. J. Physiol. 275: E321-31, 1998;Campbell, et al, J. Biol. Chem. 274: 30215-21, 1999; Firth, et al, J.Biol. Chem. 273: 2631-8, 1998). However, to date, IGFBP-3-derivedpeptides have not been shown to be sufficient for selective,high-affinity binding to any of these ligands.

This region of the molecule has also been implicated in nucleartranslocation, but the mechanism by which IGFBP-3 is internalized intotarget cells is not well understood (Schledlich, et al, J. Biol. Chem.273: 18347-52, 1998; Jaques, et al, Endocrinology 138: 1767-70, 1997). Arecently described mutant in which Domain 3 residues 228-232 of IGFBP-3have been substituted with the corresponding residues from IGFBP-1 (aclosely related protein) shows impaired binding to ALS, RXR-alpha, andplasminogen (Campbell, et al. (1998) Am. J. Physiol. 275(2 Pt1):E321-31; Firth, et al. (1998) J. Biol. Chem. 273:2631-2638). Specificproteolysis of IGFBP-3 under certain physiological conditions such aspregnancy and critical illness can lead to altered binding and releaseof its IGF ligand. The binary complex of IGFBP-3 with IGF-I or IGF-II(both growth factors bind IGFBP-3, with similar affinities) canextravasate across endothelial junctions to the intercellular milieuwhere IGFBP-3 can interact specifically with glycosaminoglycans,specific proteases, and cell-surface proteins. Research reports havereferred to the presence of a C-terminal domain in IGFBP-3 that caninhibit IGFBP-4 proteolysis (Fowlkes, et al, J. Biol. Chem. 270:27481-8, 1995; Fowlkes, et al, Endocrinology 138: 2280-5, 1997).However, the exact location of this putative protease inhibitor domainhas not yet been described. IGFBP-4 proteolysis is a key event in anumber of biological processes, including pregnancy, post-angioplastysmooth muscle cell growth, bone formation, and ovarian folliculardominance (Byun, et al, J. Clin. Endocrinol. Metab. 86: 847-54, 2001;Bayes_Genis, et al, Arterioscler. Thromb. Vasc. Biol. 21: 335-41, 2001;Miyakoshi et al, Endocrinol. 142: 2641-8, 2001; Conover,. et al,Endocrinol. 142: 2155, 2001; Rivera, et al, Biol. Reprod. 65: 102-11,2001).

It should be noted that, while IGFBP-3 is the most abundant of the IGFbinding proteins (“IGFBPs”), at least five other distinct IGFBPs havebeen identified in various tissues and body fluids. Although theseproteins bind IGFs, they originate from separate genes and have distinctamino acid sequences. Unlike IGFBP-3, other circulating IGFBPs are notsaturated with IGFs. IGFBP-3 and IGFBP-5 are the only known IGFBPs whichcan form the 150 kDa ternary complex with IGF and ALS. The IGF-bindingdomain of IGFBP-3 is thought to be in the N-terminal portion of theprotein, as N-terminal fragments of the protein isolated from serumretain IGF binding activity. However, some of the other IGFBPs have alsobeen suggested for use in combination with IGF-I as therapeutics.

In addition to its role as the major carrier protein for IGF in serum,IGFBP-3 has been recently shown to have a number of differentactivities. IGFBP-3 can bind to an as-yet unidentified molecule on thecell surface, where it can inhibit the activity of exogenously-addedIGF-I (Karas et al., 1997, J. Biol. Chem. 272(26):16514-16520). Althoughthe binding of IGFBP-3 to cell surfaces can be inhibited by heparin, theunidentified cell surface binding molecule is unlikely to be aheparin-like cell surface glycosaminoglycan, because enzymatic removalof heparin glycosaminoglycans has no effect on IGFBP-3 cell surfacebinding (Yang et al., 1996, Endocrinology 137(10):4363-4371). It is notclear if the cell surface binding molecule is the same or different thanthe IGFBP-3 receptor that was identified by Leal et al. (1997, J. Biol.Chem. 272(33):20572-20576), which is identical to the type Vtransforming growth factor-beta (TGF-β) receptor.

IGFBP-3, when used alone in in vitro assays, has also been reported topromote apoptosis. Interestingly, IGFBP-3 has been shown to promoteapoptosis in cells with and without functional type 1 IGF receptors(Nickerson et al., 1997, Biochem. Biophys. Res. Comm. 237(3):690-693;Rajah et al., 1997, J. Biol. Chem. 272(18):12181-12188). However, thereare conflicting reports as to whether apoptosis is induced by fulllength IGFBP-3 or a proteolytic fragment of IGFBP-3 (Rajah et al., ibid;Zadeh et al., 1997, Endocrinology 138(7):3069-3072). More recently, awealth of unpublished data gathered in a number of laboratories fails tosupport some of the claims made in the above publications. In in vivomodels tested to date, infused IGFBP-3 protein alone has showed mixedresults in limiting tumor growth.

U.S. Pat. No. 5,681,818 claims the administration of IGFBP-3 forcontrolling the growth of somatomedin dependent tumors in the treatmentof cancer. U.S. Pat. No. 5,840,673 also describes the indirectintracellular modulation of IGFBP-3 levels as a method for controllingtumor growth. U.S. Pat. No. 6,015,786 discloses the use of IGFBP-3complexed with mutant IGF for the treatment of IGF-dependent tumors.However, each of these patents discloses a direct in vivo effect ofadministered IGFBP-3 protein on tumor growth. All of these patentsenvisages the use of intact IGFBP-3, including its IGF-binding domain.Numerous publications (Williams, et al., Cancer Res 60(1):22-7, 2000;Perks, et al., J Cell Biochem 75(4):652-64, 1999; Maile et al.,Endocrinology 140(9):4040-5, 1999; Gill, et al., J Biol Chem272(41):25602-7, 1997) further demonstrate combined effects of IGFbinding proteins, radiation and ceramide on cultured cells. In onereport (Portera et al, Growth Hormone & IGF Research 2000, Supplement A,S49-S50, 2000) IGFBP-3 combined with CPT-11 showed additive effects in acolon cancer model both in vivo and in vitro. All of the above studieswere conducted using intact IGFBP-3, a multifunctional molecule capableof carrying IGFs (which are anti-apoptotic) to cells, while also capableof exerting IGF-independent pro-apoptotic effects of its own. Clearly itwould be of interest to separate these two activities at the molecularlevel, but molecules exhibiting a desirable subset of the activities ofintact IGFBP-3 have not been described.

IGF-I and IGFBP-3 may be purified from natural sources or produced byrecombinant means. For instance, purification of IGF-I from human serumis well known in the art (Rinderknecht et al. (1976) Proc. Natl. Acad.Sci. USA 73:2365-2369). Production of IGF-I by recombinant processes isshown in EP 0 128 733, published in December of 1984. IGFBP-3 may bepurified from natural sources using a process such as that shown inBaxter et al. (1986, Biochem. Biophys. Res. Comm. 139:1256-1261).Alternatively, IGFBP-3 may be synthesized by recombinantly as discussedin Sommer et al., pp. 715-728, MODERN CONCEPTS OF INSULIN-LIKE GROWTHFACTORS (E. M. Spencer, ed., Elsevier, N.Y., 1991). Recombinant IGFBP-3binds IGF-I in a 1:1 molar ratio.

Topical administration of IGF-I/IGFBP-3 complex to rat and pig wounds issignificantly more effective than administration of IGF-I alone (Id.).Subcutaneous administration of IGF-I/IGFBP-3 complex tohypophysectomized, ovariectomized, and normal rats, as well asintravenous administration to cynomolgus monkeys, “substantiallyprevents the hypoglycemic effects” of IGF-I administered alone (Id.).

The use of IGF/IGFBP-3 complex has been suggested for the treatment of awide variety of disorders (see, for example, U.S. Pat. Nos. 5,187,151,5,527,776, 5,407,913, 5,643,867, 5,681,818 and 5,723,441, as well asInternational Patent Applications Nos. WO 95/03817, WO 95/13823, and WO96/02565. IGF-I/IGFBP-3 complex is also under development by InsmedPharmaceuticals, Inc., as a treatment for several indications, includingdiabetes and recovery from hip fracture surgery.

For practitioners skilled in the art, the complex of IGF-I and IGFBP-3is generally considered to be a different compound, and to havedifferent biological effects, than IGFBP-3 alone.

While there are a large number of cytotoxic drugs available for thetreatment of cancer, these drugs are generally associated with a varietyof serious side effects, including alopecia, leukopenia, mucositis.Accordingly, there is a need in the art for cancer therapies that do notinduce the serious side effects associated with conventional cytotoxicchemotherapy. One method for achieving this goal is to make target cells(such as tumor cells) selectively sensitive to cytotoxic drugs, therebypermitting the effective use of such drugs at lower doses not associatedwith serious side effects. A pro-apoptotic peptide derived fromIGF-binding protein may be capable of hastening the apoptotic responseof tumor cells to chemotherapeutic and other agents (see copending U.S.application titled “Method for Use of IGF-Binding Protein for SelectiveSensitization of Target Cells In Vivo” by D. Mascarenhas, filed Sep. 18,2001).

Lifestyle changes in modern Western societies appear to have triggeredan epidemic of diseases believed to be related to longer lifespans,richer diets, modified sleep patterns, increased stress-inducing andsedentary behaviors: The possible involvement of viral co-factors(particularly Epstein-Barr virus and other herpesviruses) has also beensuspected. This constellation of diseases include cancer, cardiovasculardiseases such as atherosclerosis, autoimmune diseases such as arthritis,asthma and inflammatory bowel diseases, degenerative diseases such asosteoporosis, proliferative/inflammatory diseases such as retinopathy,and metabolic diseases such as diabetes (Grimble R F, Curr Opin ClinNutr Metab Care 5: 551-559, 2002).

A factor common to the increased incidence of most, if not all of thesediseases is the altered role of the immune system, in particular chronicinflammatory responses at the cellular level. The intracellularmolecular signatures of such responses often include activation ofglobal intracellular and extracellular regulators such as NF-kappa-B,STAT3 (Niu G et al Oncogene 21: 2000-2008, 2002), VEGF andcyclooxygenase-2 (COX-2). NF-kappa-B is a key mediator of thepro-survival induction of HIF in solid tumors (Talks K L et al Am. J.Pathol. 157: 411-421, 2000). COX-2 inhibitors are now being used togreat a variety of autoimmune indications such as arthritis, as well ascancer (Crofford L J, Curr Opin Rheumatol 14:225-30, 2002). Theanti-inflammatory agent, rapamycin (sirolimus) has been successfullyused to coat stents, with major implications for the treatment ofcardiovascular disease (Degertekin M et al, Circulation 106:1610-3,2002). Circulatory levels of C-reactive protein (CRP), a surrogatemarker for chronic inflammation, are now used as major predictors ofheart disease risk (Futterman L G and Lemberg L., Am J Crit Care 11:482-6, 2002; Libby P et al, Circulation 105:1135-43, 2002). And obesity,previously implicated as a risk factor in diabetes and heart disease,appears to provide a causal link to these diseases, as fat cells areknown to secrete pro-inflammatory cytokines (Coppack S W, Proc Nutr Soc60:349-56, 2001).

Another common molecular signature of cells playing key roles in theabove pathologies is the display of surface adhesion molecules,especially integrins. Studies have implicated alpha(v) and betaintegrins in processes as diverse as metastasis (Felding-Habermann B etal, PNAS 98: 1853-8, 2001), angiogenesis (Eliceiri B P. and Cheresh D A,Cancer J 3: S245-9, 2000), atherosclerosis (Nichols T C et al, Circ Res85:1040-5, 1999), osteoporosis (Pfaff M and Jurdic J J. Cell Sci. 114:2775-2786, 2001) and autoimmune disease. Clearly, there would be anadvantage to the use of systemic agents capable of specificallytargeting cells displaying these integrins. The advantage would beparticularly great if the same agent could also modulate levels of keyglobal pro-inflammatory regulators such as NF-kappa-B within targetcells.

IGFBP-3 and the MBD peptides of the present invention clearly exhibitboth of these desirable properties. As shown in the examples section, ina mouse tumor model (mammary 16C), tumors in animals treated withsubcutaneous daily injections of IGFBP-3 protein showed increasedsensitivity to doxorubicin (adriamycin). Post facto analysis of tumortissues showed that NF-kappa-B was downregulated 4-5-fold in tumors fromanimal treated with IGFBP-3 plus adriamycin versus those treated withadriamycin alone. In separate experiments the inventor has shown thatIGFBP-3 and MBD peptides are preferentially active upon cells expressingcertain surface integrins. In particular, antibodies to alpha(V) andcertain beta integrins can prevent nuclear uptake of MBD peptides andsubsequent co-apoptotic biochemical events. As such, IGFBP-3 and MBDpeptides present unique opportunities as agents for treating theconstellation of diseases enumerated above, as well as any otherbiological process characterized by cellular invasiveness dependent onor stimulated by alpha(v) or beta integrins and/or pro-inflammatorymolecules. An example of the latter would be the process ofcytotrophoblast implantation during fertilization (Illera M J et al,Biol. Reprod. 62: 1285-1290, 2000).

Other applications for IGFBP-3, IGFBP-derived peptides and relatedmolecules of the invention may be envisaged including modulators ordiagnostic reporters of inflammatory and invasive processes in cancermetastasis, tumor stromal activation, autoimmune diseases such assystemic lupus erythrematosis (SLE), multiple sclerosis, diabetes,ankylosing spondulitis, ulcerative colitis, Crohn's and otherinflammatory bowel disease, arthritis, asthma and allergy, boneresorptive disease, proliferative disease, wound healing,ophthalmological diseases including retinopathies, fibrotic diseases,reproductive biology, atherosclerosis and other cardiovascularindications; research tools useful in genomics- and proteomics-relatedapplications including high-throughput screening tools in drug discoveryand other research programs, reagents and vectors capable of enhancingexisting technologies for rapid expression and screening of new geneticsequences, gene therapy, diagnostics and nanotechnology applications;and in stem cell-related applications.

Numerous natural and pathological processes involve an“inflammatory-invasive” or “inflammatory-migratory” condition. Examplesinclude invasive tumors, blastocyst/cytotrophoblast implantation,atherosclerotic plaque build-up, bone turnover, joint swelling inarthritic conditions, relapsing-remitting autoimmune conditions such asmultiple sclerosis, SLE and others, proliferative retinal diseases andactivation of airway epithelium in asthmatics. A common feature of thesebiological processes is the activated state of cell types participatingin local cross-talk relevant to the disease condition. For example,invasive epithelial tumors generally include (in addition to the tumorcells themselves), activated stromal cells, microvascular epithelialcells and inflammatory immune cells. Interventions targeting any ofthese cell types might be expected to influence overall disease patternsdramatically. The inventor has unexpectedly found that IGFBP-3 andIGFBP-derived peptides preferentially trigger cell death/apoptosis insuch activated cells, compared to the same cell types withoutactivation. A corroborating observation is the dependence of theco-apoptotic effects on alpha-5 and beta-1 integrins, which are known tobe preferentially displayed by activated and migrating cells (Boles, etal, 2000, Am. J. Physiol. Lung Cell Mol. Phystol. 278: L703-L712;Laukaitis, et al, 2001,. J. Cell Biol. 153: 1427-1440) and in bonemarrow micrometastases from epithelial tumors (Putz, et al, 1999, CancerRes. 59: 241-248).

It is important to distinguish these effects from those relating toabrogation of IGF-I-dependent proliferative effects. The literature isreplete with mention of IGF-I dependent inflammatory processes such aspsoriasis. For example, U.S. Pat. No. 5,929,040 teaches the use ofinhibitors targeting the IGF-I receptor, thereby reducing skininflammation. IGFBPs can reduce signaling through this receptor bybinding and thereby sequestering IGF-I. However, the IGFBP-derivedpeptides of this invention do not bind IGF-I and are not believed toexert their effects via the IGF-I receptor.

A distinction should also be made between the present invention and U.S.Pat. No. 5,527,776 which reveals the use of intact IGFBP-3/IGF-I complexto treat subjects with immune deficiencies and anemias. The presentinvention uses non-IGF-I-binding fragments derived from IGFBP-3 alone,to treat conditions characterized by immune stimulation rather thandeficiency.

Consequently, IGFBP-3, IGPBP-derived peptides and related molecules ofthe invention may be envisaged as modulators or diagnostic reporters ofangiogenic, osteoclastogenic, atherogenic, invasive, metastatic,reproductive, arthritic, asthmatic, fibrotic, retinopathic, infective,inflammatory, neurodegenerative, stress-related, cell remodeling orimmortalization-related biological processes.

In particular, IGFBP-3-derived peptides or smaller derivative moleculesas disclosed herein may be used as protease inhibitors, metal chelators,anti-proliferative, anti-metastatic or anti-angiogenic molecules. Theymay also be useful as plasma carrier agents, facilitators of binding toextracellular matrix components, targeting agents, transporters of largeor small compounds into cells (cell internalization agents), affinitypurification tags, screening tags, transcriptional or DNA-bindingagents, cell-labeling agents, regulatory modulators, or as agentsexhibiting any combination of the above properties. In particular, suchderivative molecules may be derived from the CD74-homology domainsequence at the carboxy-terminus of IGFBP-3, and many of theseactivities have never been localized to this region of the IGFBP-3molecule before. Peptides made to sequences in this region havepreviously been shown to interfere with the binding of IGFBP-3 to anumber of its known ligands, including RXR-alpha, transferrin, ALS,plasminogen, fibrinogen and pre-kallikrein (Liu, et al, J. Biol. Chem.275: 33607-13, 2000; Weinzimer, et al, J. Clin. Endocrinol. Metab. 86:1806-13, 2001; Campbell, et al, Am. J. Physiol. 275: E321-31, 1998;Campbell, et al, J. Biol. Chem. 274: 30215-21, 1999; Firth, et al, J.Biol. Chem. 273: 2631-8, 1998). However, to date, IGFBP-3-derivedpeptides have not been shown to be sufficient for selective,high-affinity binding to any of these ligands.

The IGFBP-3-derived metal-binding domain peptides disclosed hereindiffer from previously disclosed IGFBP-3-derived molecules in a numberof important ways, including their inability to bind IGF-I, their uniqueantigenicity, and the absence of the IGFBP-3 putative death receptor(P4.33) interaction domain of IGFBP-3 (so-called “mid-region”; aminoacids 88-148). The P4.33 putative death receptor is described inInternational Patent Application No. WO 01/87238 (Genbank AccessionNumber BC031217; gi:21411477). For example, International PatentApplication No. WO 02/34916 teaches the use of point mutants of IGFBP-3in which the binding to IGF-I is impaired. However, the describedmolecules contain the mid-region of IGFBP-3 and would be expected toexert biological effects by interacting with the P4.33 putativereceptor. International Patent Application No. WO 01/87238 teaches theuse of P4.33 modulators for treating disease. The metal-binding peptidesof the present invention do not include the P4.33 putative interactiondomain (mid-region of IGFBP-3). U.S. Pat. No. 6,417,330 teaches the useof IGFBP-3 variants which are modified to be resistant to hydrolysis.Also disclosed are variant IGFBP-3s where the nuclear localizationsignal (NLS) in native IGFBP-3 is altered. Additionally,amino-terminally extended IGFBP-3s are disclosed which include a varietyof N-terminal extensions. All of these molecules differ from themetal-binding domain peptides of the present invention in two importantways: They bind IGF-I and they contain the mid-region of IGFBP-3,believed to interact with the P4.33 putative death receptor. Some recentpublications have described the use of IGFBP-3 peptides to treat cellsin culture. The only peptides found to be active on breast cancer cellsare derived from the mid-region of IGFBP-3 (McCaig, et al, 2002, Br. J.Cancer 86: 1963-1969; Perks, et al, Bioch. Biophys. Res. Comm. 294:988-994, 2002). This region is not present in the sequence of themetal-binding domain peptides of this invention.

Iron metabolism (particularly ferrous iron) offers many possibilitiesfor intervention in disease processes. For example, neoplastic cellsexpress high levels of the transferrin receptor 1 (TfR1) and internalizeiron (Fe) from tansferrin (Tf) at a very high rate. Antisense ferritinoligonucleotides inhibit growth and induce apoptosis in human breastcancer cells (Yang et al., 2002, Anticancer Res. 22(3):1513-24).Artemisinin becomes cytotoxic in the presence of ferrous iron. Sinceiron influx is high in cancer cells, artemisinin and its analogsselectively kill cancer cells under conditions that increaseintracellular iron concentrations (Singh et al., 2001, Life Sci.70(1):49-56). Iron chelators can cause apoptotic effects in cancer cells(Simonart et al., 2002, Gynecol Oncol. 85(1):95-102; Green et al., 2001,Clin. Cancer Res. 7(11):3574-9). Cancer risk is also known to beassociated with body iron stores (Kato et al., 1999, Int. J. Cancer80(5):693-8).

In addition to neoplastic conditions, many other disease states areknown to-exhibit characteristic imbalances in iron homeostasis: amongthem are Parkinson's disease (Logroscino et al., 1997, Neurology49(3):714-7), rheumatoid arthritis (Weber et al., 1988, Ann. Rheum. Dis.47(5):404-9), inflammation (Morris et al., 1995, Int. J. Biochem. Cell.Biol. 27(2):109-22) and atherosclerosis (Schmitz et al., 2001, J. Magn.Reson. Imaging 14(4):355-61), Acute iron poisoning and chronic ironoverload are well-known causes of myocardial failure. Although the exactmechanism is not known, excess iron-catalyzed free radical generation isconjectured to play a role in damaging the myocardium and alteringcardiac function (Bartfay et al., 1999, Cardiovasc. Pathol. 8(6):305-14;Parks et al., 1997, Toxicology 117(2-3):141-51). Ferrous iron can damagemitochondrial DNA (Asin et al., 2000, FEBS Lett. 480(2-3):161-4).Reperfusion injury, which occurs upon the reintroduction of blood flowto an ischemic organ, is responsible for considerable damage in heartattacks and strokes. A major cause of reperfusion injury is theiron-mediated generation of hydroxyl radical (.OH) (Horwitz et al.,1998, Proc. Natl. Acad. Sci. USA 95(9):5263-8).The use of a highlydiffusible lipophilic iron chelator secreted by Mycobacteriumtuberculosis inhibits proliferation of smooth muscle cells in culture(Rosenthal et al., 2001, Circulation 104(18):2222-7) and restenosis invivo.

Iron particles, in the form of superparamagnetic iron oxide (SPIO)particles (Ferucci, 1991, Keio J. Med. 40(4):206-14; Taupitz et al.,1993, Acta Radiol. 34(1):10-5; Mack et al., 2002, Radiology222(1):239-44) have been used to enhance contrast in magnetic resonanceimaging. More recently, these particles have been combined withalternating magnetic fields to generate local effects on iron-richcancer cells, a procedure dubbed “magnetic thermal ablation” (Hilger etal., 2002, Invest. Radiol. 37(10):580-6; Shinkei et al., 2001, Jpn. J.Cancer Res. 92(10):1138-45).

It should be noted that any reference to any patent, patent application,or publication in this Background section is not an admission that suchpatent, patent application, or publication constitutes prior art to theinstant invention.

DISCLOSURE OF THE INVENTION

The inventor has surprisingly found that IGFBP-3 or peptides containingparts of the human IGFBP-3 sequence can exhibit a large number of usefulphysical and biological properties. In particular, the inventor hasdemonstrated that the CD-74-like domain of IGFBP-3 exhibits a number ofpreviously undocumented properties, such as the ability to selectivelybind metals such as zinc and nickel. The inventor has also locatedseveral previously demonstrated activities of intact IGFBP-3 to thisregion of the molecule. The exact location of sequence boundaries forpeptides is critical to their biological activities, particularly in thecase of pro-apoptotic peptides. The inventor has also made thesurprising and provocative observation that a peptide representing a22-amino acid sequence from this region of IGFBP-3 is sufficient todirect the internalization of a covalently attached, much larger protein(green fluorescent protein) into live human embryonic kidney (HEK293)cells. The mechanism used by IGFBP-3 to enter target cells is not wellunderstood, although the existence of a cell surface receptor has beenproposed.

Additionally, the inventor has further surprisingly discovered that thecellular internalization of IGFBP-3-derived peptides is mediated bypathways involved in iron transport, particularly ferrous iron (Fe⁺⁺)transport. Accordingly, the IGFBP-3-derived peptides of the inventionare considered to be particularly useful for delivering molecules toorgans which are heavily implicated in iron metabolism, such as theheart and kidneys. These findings also indicate that reagents whichmodulate cellular uptake of iron-carrying molecules can be used tomodulate the activities of IGFBP-3 and peptides derived from it.

The inventor has also surprisingly found that the addition of a sixamino acid sequence to the carboxy-terminal end of the ‘core’ MBD 14 mersequence (QCRPSKGRKRGFCW) to create a caveolin consensus bindingsequence results in a substantial increase in nuclear translocationactivity. Peptides comprising the MBD 14 mer core sequence plus acaveolin consensus binding sequence are useful for directing nucleartranslocation of therapeutic molecules.

The present invention reveals that short peptides containing just 12-22amino acids from the C-terminal domain of IGFBP-3 can mimic the fullmolecule's co-apoptotic, cell-penetrating and metal-binding properties.These peptides (“MBD peptides”) offer an attractive alternative to theuse of full-length IGFBP-3, for the reasons listed below:

Potency: The amino-terminal domain of IGFBP-3 binds and carries IGFs incirculation, potentiating their anabolic and anti-apoptotic effects onmost cell types while the carboxy-terminal domain is believed to mediateIGF-independent effects of IGFBP-3. Thus the efficacy of this moleculeas a therapeutic may be inherently buffered by the duality of itsfunction. MBD peptides are up to 3× more active than full-length IGFBP-3in co-apoptotic assays and, unlike IGFBP-3, MBD activity is not severelyinhibited by extracellular matrix and plasma proteins;

Formulation: The central domain of IGFBP-3 is exquisitely sensitive toproteolysis. Due to the limited solubility of IGFBP-3, its low stabilityand pronounced tendency to aggregate, the development of suitableformulations and convenient routes of delivery for intact IGFBP-3 ischallenging. For example, a 1 mg/kg/day dose of IGFBP-3 (at 7 mg/ml, themaximum solubility in phosphate buffered saline) for a 70 kg adultIGFBP-3 would be 10 ml (NOT suitable for subcutaneous bolus injection),while an MBD PEPTIDE (at 35 mg/ml in PBS) given at 0.5 mg/kg/day to a 70kg adult would only 1 ml (suitable for subcutaneous bolus injection).

Stability: MBD peptides are stable to heat (95 degrees Celsius for 10minutes) and are small in size, making them more amenable to transdermalor inhalant-based delivery routes than full-length IGFBP-3.

Cost: For the IGFBP-3 molecule to be biologically active, all its 18cysteine residues must form intramolecular disulfide bonds. This makesthe production of adequate quantities of clinical grade IGFBP-3 inbacterial or yeast systems extremely challenging. Mammalian systems aretoo expensive for the industrial production of this molecule. Since thehuman IGFBP-3 cDNA was cloned in 1988, only one group has successfullyproduced gram quantities of clinical grade recombinant IGFBP-3. Theprojected selling price of a single 4 mg/kg subcutaneous dose ofIGFBP-3, based on production technologies for IGFBP-3 developed andrefined over a twelve-year period and using an industry-standard pricingratio to Cost of Goods (COG), is likely to be in the range of severalthousand dollars. Based on reimbursement and other considerations, thisprice may be prohibitive. MBD peptides, on the other hand, are expectedto be cheaper and easier to produce than IGFBP-3 using either syntheticchemical methods or highly efficient biological production systems wellknown to those skilled in the art.

Sequence alignments, combined with data from several laboratories, canprovide insights into the regions of IGFBP-3 that are likely to bestructurally autonomous while sufficient to specify the molecule'spro-apoptotic actions on tumor cells. Other studies have shown thatother sequences in this same domain are involved in nuclearlocalization, RXR-alpha-binding, and binding to serum and ECM components(21, 23-25, 35, 40). We have aligned these regions of the molecule witha CD74-homology motif seen in a disparate group of proteins across theanimal kingdom (mammalian, frog, fish, fly, nematode). For the purposesof this invention, the CD74-homology domain of IGFBP-3 is defined ascomprising approximately the 60 amino acid residues at thecarboxy-terminal end of the mature IGFBP-3 protein, or any subsetthereof.

FIG. 2 shows an alignment of selected human proteins containing the CD74motif. Conserved residues are shown in bold. Italicized residues inIGFBP-3 are required for nuclear translocation and collagen binding, butnot for IGF-I binding. Asterisks denote residues substituted in the HBDmutant of IGFBP-3, which is impaired in binding plasminogen,prekallikrein, ALS and RXR-alpha. The peptide region denoted by # issufficient to promote cell internalization when attached to greenfluorescent protein. Preliminary data further suggest that a peptiderepresenting part of this region may be sufficient to promote apoptosisin cells in culture.

Disclosed herein are methods for alleviating the symptoms of disease. Inone embodiment, an effective amount of IGF-binding protein derivativepeptide or small molecule is systemically co-administered with achemotherapeutic agent to a subject having cancer, thereby alleviatingthe symptoms of the cancer. Exogenously added IGFBP-3 sensitizes tumorsto commonly used chemotherapeutic agents, influencing both tumor'sizeand metastasis. These effects are seen with a variety of agents,including TAXOL®, 5-Fluorouracil, ADRIAMYCIN® and CPT-11, and arebelieved to reflect pro-apoptotic IGF-I-independent effects of IGFBP-3.

In another embodiment, IGF-binding protein derivative peptide or smallmolecule is systemically co-administered with other biological modifierssuch as ligands of retinoid or thyroid receptors, or antibodies capableof binding target cell molecules, to the subject with disease.

In yet another embodiment, IGF-binding protein derivative peptide orsmall molecule is administered as described in the other embodiments,but the administration occurs indirectly, using a gene sequencedelivered by a viral vector or other vehicle, or using an inducer orantagonist.

In certain aspects, the invention provides methods for alleviating thesymptoms of disease, by administering a co-administered agent togetherwith an effective amount of IGF binding protein-derived peptide or smallmolecule to a subject having the disease.

In some embodiments, the co-administered agent is a chemical agentselected from the group consisting of alkylating agents,antimetabolites, Vinca alkaloids, podophyllotoxins, antitumorantibiotics, nitrosoureas, metallic DNA modifying compounds andmicrotubule stabilizers, a biological agent selected from the groupconsisting of nutrient limitation, antibodies, vaccines, peptides,cytokines, receptor ligands and nucleic acids, or a physical agentselected from the group consisting of heat, pressure, osmolarity,acidity and radiation. Preferred co-administered agents include chemicalagents selected from the group consisting of doxorubicin, paclitaxel,methotrexate, tamoxifen, cyclophosphamide, vincristine, etoposide,streptozotocin and 5-fluorouracil.

In certain embodiments, the disease treated is cancer of the breast,prostate, colon, ovary, pancreas, stomach, esophagous or lung.

In some embodiments, the IGFBP-3-derived peptide or small molecule isadministered at about 0.001 to about 40 milligrams per kilogram totalbody weight per day (mg/kg/day).

In other aspects, the invention provides methods for alleviating thesymptoms of disease by administering an effective amount of IGF bindingprotein-derived peptide or small molecule, to a subject having thedisease.

In certain embodiments, the IGF binding protein-derived peptide is aIGFBP derived proteolysis inhibiting peptide. Such embodiments areuseful for reducing tumor invasiveness (e.g., reducing local, regional,and metastatic spread of a tumor), treating disorders associated withexcessive bone formation, and reducing or inhibiting vascularrestenosis.

In other embodiments, the IGF binding protein-derived peptide is a MBDpeptide. Such embodiments are useful for alleviating the symptoms ofdisorders mediated by chronic inflammatory responses at the cellularlevel, including cardiovascular diseases (e.g., atherosclerosis),autoimmune diseases including systemic lupus erythematosis (SLE),multiple sclerosis (MS), diabetes (especially type I diabetes),ankylosing spondulitis, ulcerative colitis, inflammatory bowel diseasesincluding Crohn's disease, arthritis (particularly rheumatoidarthritis), asthma and allergy, bone resorptive disorders,opthalmological disorders including retinopathies, and fibroticdiseases.

Additional embodiments utilize MBD peptides, and derivatives thereof,for delivery of therapeutic molecules to tissues of interest, such ascardiac, vascular, muscular, immune, liver, brain and renal tissues, andparticularly to organs which are heavily implicated in iron metabolism,such as the heart and kidneys. In such embodiments, conjugates of MBDpeptides and therapeutic molecules are administered to subjects in needthereof, such as subjects suffering from cardiac, vascular, muscular,immune, hepatic, neurologic or renal disorders, resulting inamelioration of at least one symptom the disorder sought to be treated.

The invention further provides methods of treating disorders associatedwith altered iron metabolism, such as neoplasia, Parkinson's disease,and atherosclerosis. A conjugate comprising an MBD peptide and atherapeutic molecule is administered to a subject having a disorderassociated with altered metabolism, resulting in amelioratin of at leastone symptom of the disorder.

In certain embodiments, the MBD peptide includes a caveolin consensusbinding sequence (#x#xxxx#, where ‘#’ is an aromatic amino acid) inaddition to, or overlapping with, the MBD peptide sequence. The caveolinconsensus sequence may be at the amino terminal or carboxy terminal endof the peptide. In certain preferred embodiments, the caveolin consensusbinding sequence is at the carboxy terminal end of the peptide, andoverlaps with the MBD core 14 mer sequence. Exemplary MBD peptides withcaveolin consensus binding sequences include peptides comprising thesequence QCRPSKGRKRGFCWAVDKYG or KKGFYKKKQCRPSKGRKRGFCWAVDKYG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B shows sequences IGFBP-3 in single letter amino acid code.FIG. 1A shows the amino acid sequences of native human IGFBP-3 (Ala₅allelic variant). FIG. 1B shows [N109D]-hIGFBP-3 derivative (Ala₅allelic variant).

FIG. 2 shows an amino acid sequence alignment of selected human proteinscontaining the CD74 motif.

FIG. 3 depicts the results of the experiment described in Example 1.

FIGS. 4A-4B depicts the results of the experiment described in Example2.

FIGS. 5A-5B shows results of IMAC purification of IGFBP-3 using Ni⁺⁺ andZn⁺⁺ IMAC. Panels a and b (FIGS. 5A and 5B) show SDS-PAGE analysis ofsamples from Ni⁺⁺ and Zn⁺⁺ IMAC, respectively. FT indicates columnflowthrough; W indicates wash; 50 indicates 50 mM imidazole wash; 60indicates 60 mM imidazole wash; E indicates 1 M imidazole elutionbuffer; S indicates 1 M EDTA stripping buffer.

FIG. 6 shows a scheme for production of IGFBP-3 domains using fusionproteins. The lower panel shows SDS-PAGE analysis of purification. Lane1 shows crude extract digested with 3C proteinase (10:1 dilution); lane2 shows after Phenyl-Sepharose HIC Chromatography; and lane 3 showsafter nickel metal affinity chromatography.

FIG. 7 shows fluorescence of cells after incubation with peptide-gfpfusions. a is cells alone (no gfp added); b is cells plusSYGRKKRRQRRRAHQNSQT-gfp; c is cells KKGHAKDSQRYKVDYESQS-gfp; d iscells+KKGFYKKKQCRPSKGRKRGFCW-gfp.

FIGS. 8A-8C shows graphs summarizing the results of co-apoptosis assaysdescribed in Example 8.

FIGS. 9A-9B shows the results of the cell internalization experimentsdescribed in Example 9.

FIGS. 10A-10B shows the results of the antigenic profiling studiesdescribed in Example 11.

FIGS. 11A-11C summarizes the results of experiments which confirm theIGF-independence of the co-apoptotic activity of both IGFBP-3 and MDB2.

FIGS. 12A-12B summarizes the results of experiments showing that theco-apoptotic activity of MDB2 is integrin dependent.

FIGS. 13A-13B summarizes the results of experiments showing that theco-apoptotic activity of MDB2 is sequence specific.

FIGS. 14A-14B summarizes the results of experiments showing that IGFBP-3stimulates bax-alpha expression, and that bax-alpha expression iscorrelated with caspase-3 activity.

FIG. 15 summarizes the results of experiments showing that theco-apoptotic activity of MDB peptides is dependent on PI3K/ILK, but notMAPK, signal transduction pathways.

FIGS. 16A-16B summarizes the results of experiments showing that MDBpeptides sensitize carcinoma cells to low doses of cytotoxicchemotherapy agents.

FIG. 17 summarizes the results of the experiment described in Example19. Open squares indicate data from IGFBP-3 incubated in saline; opencircles indicate data from IGFBP-3 incubated with ferrous chloride;closed circles indicate data from IGFBP-3 incubated with IGF-I andferrous chloride; open triangles indicate data from IGFBP-3 incubatedwith ferric chloride; and open diamonds indicate data from IGFBP-3incubated with zinc chloride.

FIG. 18 summarizes the results of the experiment described in Example20, measuring the effects of preincubation of MBD-GFP32 with certainmetals on nuclear translocation. Each bar represents the average ofthree measurements.

FIG. 19 summarizes the result of an experiment described in Example 21,measuring the effects of nystatin and chlorpromazine on nucleartranslocation of MBD peptides. Each bar indicates the average of threeplates.

FIG. 20 summarizes the result of an experiment described in Example 21,measuring the effects of nystatin and chlorpromazine on the associationof MBD peptides with α5 integrin. Data from cells treated withchlorpromazine is indicated by open diamonds. Data from cells treatedwith nystatin is indicated by open circles. Data from control(untreated) cells is indicated by open squares.

FIG. 21 summarizes the result of an experiment described in Example 21,measuring the effects of nystatin and chlorpromazine on the associationof MBD peptides with the transferrin receptor. Data from cells treatedwith chlorpromazine is indicated by open diamonds. Data from cellstreated with nystatin is indicated by open circles. Data from control(untreated) cells is indicated by open squares.

FIG. 22 summarizes the results of an experiment investigating thedistribution of MBD-GFP conjugates. For each tissue, data is presented,left to right, for saline, MBD-GFP32, and MBD-GFP37. Each bar representsthe average of the measurements from three animals.

FIG. 23 summarizes the results of an experiment investigating thedistribution of MBD-GFP conjugates following 18 days of pretreatmentwith mMBD7. For each tissue, data is presented, left to right, forsaline, MBD-GFP32, and MBD-GFP37. Each bar represents the average of themeasurements from three animals.

FIG. 24 summarizes the results of the experiment described in Example21, measuring nuclear translocation of biotinylated peptides MBD9,MBD20, and MBD21 complexed with streptavidin horse radish peroxidase(HRP). Column 1 designates results from control (no peptide); column 2designates results from MBD9/HRP conjugate; column 3 designates resultsfrom MBD20/HRP conjugate; column 4 designates results from MBD21/HRPconjugate.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention relates to a number of new peptides (and small moleculeswhich mimic the structure of the peptides) which have a variety ofuseful properties, including metal-binding, extracellular matrix (ECM)binding, directing cell internalization, inhibiting protease activity,modulation of transcription, apoptosis promotion, angiogenesisinhibition, anti-inflammatory activities, as well as utilities for cellimaging and expression tagging.

Disclosed herein are new methods for the treatment of disease. Thevarious activities of the peptides of the invention may be utilized totreat a range of disorders, including cancer, autoimmune disease,cardiovascular indications, arthritis, asthma, allergy, reproductiveindications, retinal proliferative disease, bone disease, inflammatorydisease, inflammatory bowel disease, and fibrotic disease.

In certain embodiments, an effective amount of pro-apoptotic peptide anda co-administered agent are systemically administered to a subjectsuffering from cancer, thereby alleviating the symptoms of the cancer. Awide variety of malignancies may be treated using the methods of theinvention, including breast, prostate, colon, ovarian, pancreatic,gastric and lung cancer. The co-administered agent is typically ancytotoxic chemotherapy agent, such as doxorubicin, paclitaxel,methotrexate, tamoxifen, cyclophosphamide, vincristine, etoposide,streptozotocin and 5-fluorouracil

DEFINITIONS

As used herein, the terms “IGF-binding protein” and “IGFBP” refer tonatural and derivative molecules based on any of the six humaninsulin-like growth factor binding proteins 1 through 6. “Derivativepeptide or small molecule” refers to peptides or peptidomimetics, thatretain, or mimic those structural properties of IGFBPs that are relevantto the instant invention. Derivative peptides herein comprise less thanthe full length sequence of IGFBP-3. As used herein, a peptide or smallmolecule is “derived from” an IGFBP if its sequence or structure isidentical or homologous to the IGFBP.

“CD74-homology domain peptides or small molecules” means derivativepeptides or small molecules containing part of the carboxyterminal60-amino acid sequence of IGFBP-3.

“Metal-binding domain peptide” or “MBD peptide” means an IGFBP-derivedpeptide or polypeptide from about 12 to about 60 amino acids long,preferably from about 13 to 40 amino acids long, comprising a segment ofthe CD-74-homology domain sequence in the carboxy-terminal 60-aminoacids of IGFBP-3, comprising the sequence CRPSKGRKRGFC and exhibitingmetal-binding properties, but differing from intact IGFBP-3 byexhibiting distinct antigenic properties, lacking IGF-I-bindingproperties, and lacking the mid-region sequences (amino acids 88-148 ofIGFBP-3 sequence). For example, the peptide GFYKKKQCRPSKGRKRGFCW is anexample of a metal-binding domain peptide. It binds metal ions but notIGF-I, and polyclonal antibodies raised to this peptide do notsubstantially cross-react with intact IGFBP-3, and vice versa.

“Extended metal-binding domain peptides” are metal-binding domainpeptides linked to additional residues' differing from the naturalIGFBP-3 sequence. For example, extensions such as the tripeptideasparagine-glycine-arginine (NGR), or a large protein sequence may beadded for pharmacokinetic targeting purposes, or for the preparation ofconjugates with other molecules such as lipids and nucleic acids(“extended metal-binding domain peptide conjugates”).

“Modified metal-domain peptides” are metal-binding domain peptides orextended metal-binding domain peptides in which the natural amino acidsequence has been modified, such modifications including conservativesubstitutions for the natural amino acid residue at any position in thesequence, alteration of phosphorylation, acetylation, glycosylation orother chemical status found to occur at the corresponding sequenceposition of IGFBP-3 in the natural context, substitution of D- forL-amino acids in the sequence, or modification of chain backbonechemistry, such as protein-nucleic-acid (PNA).

“Core metal-binding-domain peptides” are peptides less than 14 aminoacids long comprising the core 12-mer sequence, CRPSKGRKRGFC. Forexample, CRPSKGRKRGFC, QCRPSKGRKRGFC and CRPSKGRKRGFCW are coremetal-binding domain peptides.

“Extended core metal-binding domain peptides” are extended metal-bindingdomain peptides comprising the core 12-mer sequence CRPSKGRKRGFC, butnot comprising the following natural IGFBP-3 14-mer sequence:QCRPSKGRKRGFCW.

“Modified core metal-binding peptides” are modified metal-binding domainpeptides comprising the core 12-mer sequence CRPSKGRKRGFC, but notcomprising the following natural IGFBP-3 14-mer sequence:QCRPSKGRKRGFCW.

“Retro metal-binding domain peptides” are derivatives of metal-bindingdomain peptides containing either D- or L-amino acids in reverse order.

“Cell internalization peptides” means a peptide or other proteinaceousmolecule, or mutant or other derivative thereof, comprising the sequenceKKGFYKKKQCRPSKGRKRGFCW or part thereof, which is present in theCD74-homology domain of IGFBP-3.

“Plasma circulatory peptide” means CD74-homology domain peptideretaining some or all of the circulatory plasma protein bindingcharacteristics of IGFBP-3. Binding to plasminogen, transferrin,kallilrein, acid-labile subunit, or fibrinogen are examples in thiscategory.

“ECM-binding, peptide” means CD74-homology domain peptide retaining someor all of the extracellular matrix component binding characteristics ofIGFBP-3. Binding to heparin, collagen and cell surface components areexamples in this category.

“Protease inhibitor peptide” means CD74-homology domain peptideretaining some or all of the protease inhibitor characteristics ofIGFBP-3. In particular this refers to inhibition of cysteine proteases,serine proteases and metalloproteases. “Expression vector tag” means anyCD74-homology domain peptide sequence included in a gene expressionvector wherein the properties of IGFBP-3 retained in the vector tag areinstrumental in facilitating the use of the vector for research,high-throughput screening or other applications well recognized in theart.

“Pro-apoptotic peptide” means a CD74-homology domain peptide retainingsome or all of the pro-apoptotic characteristics of IGFBP-3 but not itsIGF-binding characteristics. In one embodiment, “core” pro-apoptoticpeptides contain the first sequence QCRPSKGRKRGFC, but not the secondsequence KKGFYKKK, which lies almost immediately adjacent to the firstsequence in the intact IGFBP-3 molecule. As is known to those skilled inthe art, it is possible to make sequence changes to any protein orpeptide sequence without substantially modifying its properties. Thus,peptide sequences exhibiting comparable biological activity and at least80% sequence homology, more preferably 85, 90, 95, 98, or 99% sequencehomology, to any of the above sequences are also covered by the abovedefinitions.

The term “co-administered agent”, as used herein, refers to a chemicalagent; a biological agent such as an antibody, vaccine, nutrient,cytokine, nucleic acid or receptor ligand such as growth factor,retinoid or thyroid receptor ligand; and a physical agent, such asradiation, acidity and heat. Co-administered agents preferably have ananti-tumor activity when administered in the absence of IGFBP.

“Chemical agents” include all common chemotherapeutic agents such asalkylating agents (e.g. busulfan, cyclophosphamide, ifosfamide),antimetabolites (e.g. Ara-C, 5-fluorouracil, methotrexate), Vincaalkaloids (e.g. vinblastine, vincristine), podophyllotoxins (e.g. VM-26,etoposide), antibiotics (e.g. bleomycin, doxorubicin/ADRIAMYCIN®),nitrosoureas (e.g. BCNU, streptozotocin), and metallic DNA modifyingcompounds (e.g. carboplatin, cisplatin), and microtubule stabilizers(e.g., paclitaxel/TAXOL®). Chemical agents also include chemicalcompounds that directly affect a targeted receptor by reducing levels ofthe cognate ligand, by acting on the targeted receptor or acting on thesignaling pathway of the targeted receptor. For example, the thyroidaxis may be indirectly manipulated via antagonists such as thyroid axisantagonists. As an example, the term “thyroid axis antagonist” refers toa compound which acts to decrease thyroid hormone activity in a subject.Thyroid axis antagonists include 6-n-propyl-2-thiouracil(propylthiouracil or PTU), methimazole, carbimazole, and other compoundsknown to the art to reduce thyrotropic hormones, thyroid hormones, orthyroid receptor signaling.

The term “treatment regimen” refers to a course of therapy. Treatmentregimens may utilize a single agent such as a single chemical agent, butmore typically involve two or more different agents (e.g., combinationtherapy with multiple different cytotoxic chemotherapy agents), and mayinvolve two or more different types of agents (e.g., administration of achemical agent such as paclitaxel in combination with a physical agentsuch as ionizing radiation). Treatment regimen may also refer tonutritional, stress or exercise regimen.

The term “alleviating”, as used herein, refers to an improvement,lessening, stabilization, or diminution of a symptom of a disease.“Alleviating” also includes slowing or halting progression of a symptom.For example, alleviating a symptom of cancer includes slowing orstabilizing tumor growth, reducing tumor size, or eliminating the tumorentirely.

The term “subject”, as used herein, refers to a vertebrate individual,including avian and mammalian individuals, and more particularly tosport animals (e.g., dogs, cats, and the like), agricultural animals(e.g., cows, horses, sheep, and the like), and primates (e.g., humans).

Sequence “identity” and “homology”, as referred to herein, can bedetermined using BLAST (Altschul, et al., 1990, J. Mol. Biol.215(3):403-410), particularly BLASTP 2 as implemented by the NationalCenter for Biotechnology Information (NCBI), using default parameters(e.g., Matrix 0 BLOSUM62, gap open and extension penalties of 11 and 1,respectively, gap x_dropoff 50 and wordsize 3). Unless referred to as“consecutive” amino acids, a sequence optionally can contain areasonable number of gaps or insertions that improve alignment.

As used herein, the term “conjugate” includes both covalent andnoncovalent conjugates between a peptide and second molecule (such as atranscriptional modulator or a therapeutic molecule). Noncovalentconjugates may be created by using a binding pair, such as biotin andavidin or streptavidin or an antibody (including Fab fragments, scFv,and other antibody fragments/modifications) and its cognate antigen.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

IGF-Binding Protein Derivative Peptide and Small Molecule Compositions

IGF-binding protein derivative peptide or small molecule for use inaccordance with the instant inventive methods may be derived from anyspecies, although species-matched IGF-binding protein (i.e., IGF-bindingprotein derivative peptide or small molecule based on the nativesequence from the same species as the subject to which the IGF-bindingprotein derivative peptide or small molecule is to be administered) ispreferred (e.g., when the IGF-binding protein derivative peptide orsmall molecule is intended to be administered to a human subject, it ispreferred that the IGF-binding protein derivative peptide or smallmolecule be derived from a human IGFBP). IGF-binding protein derivativepeptide or small molecule for use in the instant invention isuncomplexed IGF-binding protein derivative peptide or small molecule,that is, administered in the absence of IGF (e.g., not administered asIGF-I complex), and is preferably administered without any IGF protein.Preferably, IGF-binding protein derivative peptides or small moleculesare derived from IGFBP-3.

One of the naturally occurring protein sequences for IGFBP-3 is shown inFIG. 1. Human IGFBP-3 is found in two naturally occurring allelicvariants; alanine may be found at position 5 of the mature protein(shown in FIG. 1 a), or alternately glycine may be found in thisposition. Additionally, other variants of IGFBP-3 may be created. Forexample, [N109D]-IGFBP-3 is a derivative of IGFBP-3 that has an aminoacid sequence alteration at position 109 of the mature sequence butbehaves very similarly to wild type IGFBP-3 in most assays tested todate. Point mutant derivatives also include mutants selectivelydebilitated in their ability to bind IGF-I, IGF-II, or any other knownligands of IGFBPs. For example, it has been shown that point mutationsat positions corresponding to one or more of the conserved orsemi-conserved residues Va₄₉, Tyr₅₀, Pro₆₂, Lys₆₈, Pro₆₉, Leu₇₀, Ala₇₂,Leu₇₃, and Leu₇₄ of IGFBP-5 may be debilitated in IGF-I binding. Many ofthese residues are well-conserved in the other IGF-binding proteins aswell. Mutations at positions 228 and 230 of the mature sequence ofIGFBP-3 are believed to affect nuclear translocation and binding toextracellular matrix proteins such as collagen.

Deletion mutants of IGFBP-3 or peptide derivatives based on parts of theIGFBP-3 sequence, may also be used as the template for design ofderivative peptides and small molecules. The IGFBP-3 molecule consistsof 264 amino acids and has three major structural domains. Thecysteine-rich amino terminal domain (roughly the first 100 amino acidsof the mature sequence) is known to be essential for high-affinitybinding of IGFs. The middle domain (about 80 amino acids) has nocysteine residues, and is very susceptible to proteases. It may alsoplay a role in binding specific cellular receptors. The carboxy-terminaldomain (about 80 amino acids) is also cysteine-rich and containssequences essential for binding extracellular matrix molecules such asheparin and collagen, serum molecules such as ALS, plasminogen, andfibrinogen, nuclear receptors such as RXR, and importin. Methods fornucleic acid manipulation, protein expression and protein purificationfor obtaining deletion or point mutants are known in the art.

Once a domain of IGFBP-3 has been defined by point mutation or deletionanalysis as necessary and sufficient for a particular biologicalactivity, such as the sensitization of target cells, it is possible todesign smaller molecules, such as peptides, consisting of part of theIGFBP sequence. For example, one- or more of the sequences (SEQ ID NO:1)(H2N)...DKKGFYKKKQCRPSKGRKRGFCW...(COOH); (SEQ ID NO:2)(H2N)...GFYKKKQCRPSKGRKRGFCW...(COOH); (SEQ ID NO:3)(H2N)...QCRPSKGRKRGFCW...(COOH); and (SEQ ID NO:4)(H2N)...CRPSKGRKRGFC...(COOH)may be sufficient to mimic some of the biological effects of IGFBP-3,although certain embodiments of the invention may exclude peptides whichcomprise or consist of the sequences DKKGFYKKKQCRPSKGRKRGFCW andQCRPSKGRKRGFCW.

Although the three-dimensional structure of IGFBP-3 is not known, thestructure of CD74 invariant chain, which shares considerable homologywith the relevant region of the IGFBP-3 molecule, has been described(Ghosh, et al, Nature 378: 457-462, 1995) Peptidomimetic moleculesderived from an IGFBP (preferably IGFBP-3) sequence may be created byreference to the three-dimensional structure of CD74 invariant chain,using techniques known in the art. Any of these derivative molecules maybe assayed for the desired biological activities, including the abilityto sensitize target cells to chemical treatments. Based on the resultsof these assays, a small number of IGFBP-3 mutants or derivatives withaltered characteristics may be selected for clinical testing in thecontext of human disease.

IGFBP derived protease inhibitor peptides and peptidomimetics are alsocontemplated within the present invention. Such peptides andpeptidomimetics are useful as inhibitors of proteolytic activity thatcleaves IGFBP-4. As noted above, cleavage of IGFBP-4 by certainproteases results in an effective increase in IGF-I activity, which isimportant in a number of different processes, including proliferation ofvascular smooth muscle cells. Accordingly, protease inhibitor peptidesare useful for the inhibition of vascular restenosis, particularlyarterial restenosis following angioplasty (with or without stentimplantation) and coronary artery bypass surgery. Additionally,proteolysis of IGFBP-4 is important in promoting bone formation.Accordingly, proteolysis inhibiting peptides are also useful forinhibiting bone formation in indication which involve excessive boneformation. Proteolysis inhibiting peptides are also useful forinhibition of cysteine proteases such as Cathepsin B andmetalloproteases such as MMP-2 and MMP-9, which are known to beassociated with tumor invasiveness. Accordingly, the invention providesmethods of reducing tumor invasiveness (e.g., reducing local, regional,and metastatic spread of a tumor) by administering an IGFBP derivedproteolysis inhibiting peptide.

Also provided are peptides incorporating the MBD peptide core 14 mersequence plus a caveolin consensus binding sequence. The caveolinconsensus sequence may be located upstream (i.e., towards the aminoterminus) or downstream (i.e., towards the carboxy terminus) of thepeptide, and may overlap with the MBD core 14 mer sequence. Exemplaryembodiments include the peptides MBD20 (QCRPSKGRKRGFCWAVDKYG) and MBD21(KKGFYKKKQCRPSKGRKRGFCWAVDKYG).

IGF-Binding Protein Derivative Peptide Production

The IGF-binding-protein or derivative is normally produced byrecombinant methods, which allow the production of all possible variantsin IGFBP sequence. Techniques for the manipulation of recombinant DNAare well known in the art, as are techniques for recombinant productionof proteins (see, for example, in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, Vols. 1-3 (Cold Spring Harbor Laboratory Press, 2ed., (1989); or F. Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Green Publishing and Wiley-Interscience: New York, 1987) andperiodic updates). Derivative peptides or small molecules of knowncomposition may also be produced by chemical synthesis using methodswell known in the art.

Nucleic acid vectors containing sequences encoding IGF-binding proteinderivative peptides or small molecules may utilize the varied propertiesof these molecules to facilitate the implementation of research,high-throughput screening or other genomics- and proteomics-relatedtechnologies. In particular the metal-binding characteristics of someCD74-like peptides may aid in rapid affinity purification of expressedgene products from crude extracts using immobilized metal affinitychromatography (IMAC) resins such as His-Bind (Novagen Inc., Madison,Wis.), Nickel-NTA (Qiagen Inc., Carlsbad, Calif.) and Talon (ClontechInc., Palo Alto, Calif.) resins, using the protocols recommended by themanufacturers of such resins. Likewise, the protease inhibitorcharacteristics of some CD74-like peptides may aid in expressed geneproduct stability during purification. The cell internalizationproperties of some CD74-like peptides may aid in rapid screening ofcertain gene products in mammalian cells, particularly in those caseswhere transport to the cell nucleus may facilitate screening ofbiological properties. The use of selective proteolytic cleavage siteswithin IGF-binding protein-derived sequences in vectors can aid in therecovery of properly folded domains of IGFBPs or peptide derivativesthereof. The use of human rhinovirus 3C proteinase for effectingpost-expression cleavage of gene products containing such sequences isparticularly recommended (ref).

Preferably, the IGF-binding protein or derivative is produced using abacterial cell strain as the recombinant host cell. An expressionconstruct (ie., a DNA sequence comprising a sequence encoding thedesired IGF-binding protein or derivative operably linked to thenecessary DNA sequences for proper expression in the host cell, such asa promoter and/or enhancer elements at the 5′ end of the construct andterminator elements in the 3′ end of the construct) is introduced intothe host cell. The DNA sequence encoding the IGF-binding protein orderivative may optionally linked to a sequence coding another protein (a“fusion partner”), to form a fusion protein. Preferably, the DNA,sequence encoding the IGF-binding protein or derivative is linked to asequence encoding a fusion partner as described in U.S. Pat. No.5,914,254. The expression construct may be an extrachromosomalconstruct, such as a plasmid or cosmid, or it may be integrated into thechromosome of the host cell, for example as described in U.S. Pat. No.5,861,273.

Conjugates and Fusions Incorporating IGFBP-Derived Peptides

As disclosed herein, the peptide KKGFYKKKQCRPSKGRKRGFCW is capable ofdirecting cellular internalization of an unrelated protein, and isparticularly useful for directing cellular internalization into cardiacand renal tissues. Accordingly, the invention provides fusions and/orconjugates of IGFBP-derived internalization peptides and small moleculeswith molecules which are desired to be internalized into cells. Thefusion partner molecules may be polypeptides, nucleic acids, or smallmolecules which are not normally internalized (e.g., because of largesize, hydrophilicity, etc.). As will be apparent to one of skill in theart, such fusions/conjugates will be useful in a number of differentareas, including pharmaceuticals (to promote internalization oftherapeutic molecules which do not normally become internalized), genetherapy (to promote internalization of gene therapy constructs), andresearch (allowing ‘marking’ of cells with an internalized markerprotein). Preferred IGFBP-derived internalization promoting peptides arepeptides comprising the sequence KKGFYKKKQCRPSKGRKRGFCW or a sequencehaving at least 80, 85, 90, 95, 98, or 99% homology to said sequence,wherein the peptide does not comprise the full sequence of IGFBP-3.Fusions of IGFBP-derived internalization peptides and polypeptides arepreferably made by creation of a DNA construct encoding the fusionprotein, but such fusions may also be made by chemical ligation of theinternalization peptide and the polypeptide of interest. Conjugates ofIGFBP-derived internalization peptides and nucleic acids or smallmolecules can be made using chemical crosslinking technology known inthe art. Preferably, the conjugate is produced using aheterobifunctional crosslinker to avoid production of multimers of theinternalization peptide.

Conjugates of an IGFBP-derived internalization promoting peptide andtranscriptional modulators (e.g., transcription factors) are provided bythe invention. Nearly all transcription factors are intracellularproteins which are not normally capable of being internalized from theextracellular milieu, making them unsuitable as pharmaceutical agents intheir native form. However, when fused or conjugated with anIGFBP-derived internalization promoting peptide, the transcriptionfactor can be internalized and affect cellular transcription. Forexample, T-bet (Szabo et al., 2000, Cell 100(6):655-69), a transcriptionfactor that appears to commit T lymphocytes to the T_(h1) lineage, canbe fused to a an IGFBP-derived internalization promoting peptide tocreate a molecule useful in immunomodulation.

Conjugates of cell internalization peptides and therapeutic moleculesare also provided. Cell internalization peptides may be conjugated withany therapeutic molecule which is desired to be delivered to theinterior of a cell, including antisense oligonucleotides andpolynucleotide constructs (e.g., encoding therapeutic molecules such asgrowth factors and the like).

Peptides comprising cell internalization peptide (e.g., the MBD core 14mer) and a caveolin consensus binding sequence (MBD/caveolin peptides)may also be incorporated into conjugates. MBD/caveolin peptides may beconjugated with any therapeutic molecule that is desired to be deliveredto the interior of a cell, including antisense oligonucleotides andpolynucleotide constructs (e.g., encoding therapeutic molecules such asgrowth factors and the like).

Also provided are fusion/conjugate molecules comprising a marker moietyand an IGFBP-derived internalization promoting peptide. Marker moietiesuseful in such fusion/conjugate molecules include proteins such greenfluorescent protein, luciferase, and other proteins which can bedetected by virtue of an enzymatic activity (such as alkalinephosphatase, β-galactosidase, and the like), as well as ‘expression tag’moieties which can be detected by a secondary detection system such asspecific antibodies. Expression tag moieties are well known, and includepeptides derived from myc and other proteins. Due to the localization ofIGFBP-3 to the nucleus of rapidly dividing cells, fusion/conjugatemolecules comprising IGFBP-derived internalization promoting peptidescomprising the sequence KKGFYKKKQCRPSKGRKRGFCW or a sequence having atleast 80, 85, 90, 95, 98, or 99% homology to said sequence, are believedto be particularly useful for cell labeling uses and diagnostic uses.Other contemplated uses of such fusion/conjugate molecules comprising anIGFBP-derived internalization promoting peptide include pharmacokineticstudies of pharmaceutical molecules incorporating an IGFBP-derivedinternalization promoting peptide.

Fusion/conjugate molecules comprising a ECM-binding peptide are alsoprovided. The fusion/conjugate molecules are targeted to theextracellular matrix through the ECM binding peptide. A peptide orpeptidomimetic derived from the CD74 homology domain of IGFBP may beconjugated or produced as a fusion with a different polypeptide or witha small molecule. In alternative embodiments, the ECM binding peptidemay be conjugated with or inserted into the surface of a liposome (orother encapsulation formulation) to target the combination to theextracellular matrix. While not wishing to be bound by any particulartheory, the inventor believes that IGFBP derived ECM binding peptidesare useful for both ECM binding as well as targeted release of thefusion/conjugate partner at sites in which IGFBP-cleaving protease ispresent.

As disclosed in Example 3, the inventor has discovered the presence of ametal-binding motif in the IGFBP-3 molecule, allowing practical recoveryof domains containing this motif. The IGFBP-derived peptidesDKKGFYKKKQCRPSKGRKRGFCW and QCRPSKGRKRGFCW each bound to an affinitycolumn loaded with nickel. Such metal binding properties may be used forpurification of desired peptides from complex mixtures (such asbacterial cell lysates). Typically, a DNA sequence encoding eitherpeptide (or homologue thereof having metal binding activity and at least80, 85, 90, 95, 98, or 99% sequence homology) is fused to a DNA sequenceencoding a polypeptide of interest, wherein the peptide does notcomprise the full sequence of IGFBP-3. The sequence encoding the metalbinding peptide may be fused to the 5′ or 3′ end of the DNA sequenceencoding the polypeptide of interest, and may even be inserted withinthe sequence of interest (although this is less preferred). Preferably,DNA encoding for a recognition site for an endoprotease is insertedbetween the sequence encoding the metal binding peptide and thepolypeptide of interest, to allow removal of the metal binding peptide.Useful protease recognition sites include the recognition site of humanrhinovirus 3C protease, enterokinase, Factor Xa, and ubiquitin (therecognition site of ubiquitinase). The DNA encoding the fusionpolypeptide (comprising the IGFBP-derived metal binding peptide and thepolypeptide of interest, and optionally the protease recognition site,)is then inserted into any convenient expression vector comprising theDNA sequences necessary for transcription and translation of the encodedfusion polypeptide. The DNA expression construct is transformed into arecombinant host, such as E. coli, or S. cerevisiae, and recovered usingstandard methods known in the art. The fusion polypeptide can then bepurified using an affinity column loaded with a divalent cation such aszinc or nickel, as is well known in the art. If the fusion polypeptidecomprises a protease recognition site, the cognate protease may be usedto cleave the metal binding peptide from the polypeptide of interest atan appropriate point in the purification process.

Also disclosed are methods for generating properly folded sub-domains ofIGFBP-3. The practical significance of this approach in the case ofIGFBP-3 is that numerous unsuccessful attempts have already been made,in a number of laboratories, to express truncated segments of IGFBP-3 inproperly folded form. To date, these have proved relatively unsuccessfulin generating such properly folded molecules as a major percentage ofthe total expressed product. By generating the intact molecule andcleaving it post facto, it is possible to generate folded domains withsubstantially higher efficiencies.

As demonstrated in Example 5, properly folded sub-domains of IGFBP-3 maybe produced by engineering target sites for a specific protease atstrategic locations in the IGFBP-3 sequence, expressing the construct,and cleaving the expressed protein with the cognate protease. As will beapparent to one of skill in the art, this method is useful forproduction of both naturally occurring and variant sub-domains ofIGFBP-3. The techniques for carrying out this method are well known inthe art, and involve recombinant DNA engineering to insert proteaserecognition sites into the IGFBP-3 sequence. A variety of differentprotease recognition sites are known, as discussed above, and anyconvenient protease recognition site may be used, so long as the site isnot already present in the IGFBP-3 sequence. The recognition site of the3C protease of human rhinovirus is a preferred protease recognitionsite. The construct containing the DNA sequence encoding IGFBP-3 withinserted protease sites is then inserted into an appropriate expressionvector comprising the signals necessary for transcription andtranslation of the IGFBP-3 construct sequence. The IGFBP-3 is thenproduced by transformation of the expression construct into anappropriate recombinant host and expressed. Preferably, the IGFBP-3 ispurified from the recombinant expression system, refolded (ifnecessary), then cleaved to render properly folded sub-domains ofIGFBP-3.

Therapeutic Administration

An IGF-binding protein derivative peptide or small molecule, incombination with agents or treatment regimens causing cellular damage orstress, may be used to treat any disease or disorder for which cytotoxicor cytostatic therapy is indicated, including cancer, preferablycarcinomas of the breast, prostate, colon and lung, hyperproliferativedisorders including proliferation of inflammatory or otherimmune-related cells, and arterial restenosis (e.g., after angioplastyand/or coronary artery bypass surgery). In certain embodiments, theco-administered agent is a chemotherapy agent (e.g., paclitaxel,vincristine, and the like), which may be conjugated to an IGFBP-derivedECM binding peptide to provide targeting and localization.

The invention also provides methods of methods of reducing tumorinvasiveness (e.g., reducing local, regional, and metastatic spread of atumor), methods of reducing bone formation, particularly in disordersassociated with excessive bone formation, and methods of reducing orinhibiting vascular restenosis by administering an IGFBP derivedproteolysis inhibiting peptide.

The invention further provides methods of treating (e.g., alleviatingthe symptoms of) disorders including cardiovascular diseases includingatherosclerosis, autoimmune diseases including systemic lupuserythematosis (SLE), multiple sclerosis (MS), diabetes (especially typeI diabetes), ankylosing spondulitis, ulcerative colitis, inflammatorybowel diseases including Crohn's disease, arthritis (particularlyrheumatoid arthritis), asthma and allergy, bone resorptive disorders,opthalmological disorders including retinopathies, and fibroticdiseases. As discussed in the Background, supra, these disorders aremediated by chronic inflammatory responses at the cellular level. Inaccordance with the invention, these disorders are treated byadministration of an effective amount of a MBD peptide of the invention.

Also provided are therapeutic methods comprising administration ofcompositions comprising fusions or conjugates of transcriptionalmodulators and IGFBP-derived internalization peptides. In certainembodiments, the IGFBP-derived internalization peptide further comprisesa caveolin consensus binding sequence. In some embodiments, the fusionor conjugate comprises an IGFBP-derived internalization peptide and atranscription factor such as T-bet. T-bet containing conjugates areuseful for immunomodulation, shifting or biasing an immune responsetowards a T_(h1) response, which can alleviate symptoms of disorderssuch as allergy, auto immune disease such as rheumatoid arthritis, andother T_(h2) mediated disorders.

Further provided are methods of treating cardiac, vascular, muscular,immune, liver, brain and renal disorders, comprising administeringconjugates of a therapeutic molecule and an IGFBP-derivedinternalization peptide. In certain embodiments, the IGFBP-derivedinternalization peptide further comprises a caveolin consensus bindingsequence. These embodiments exploit the differential uptake ofIGFBP-derived internalization peptides into cardiac, skeletal muscle,splenic, and renal tissue, delivering the therapeutic molecule tointerior of cells in the target tissue. The therapeutic molecule may beany molecule which requires cellular internalization for activity in thetarget organ. One class of preferred therapeutic molecules for use inthese embodiments is antisense oligonucleotides. Small inhibitory RNAs(siRNAs) is also a preferred class of therapeutic molecules. Fortreatment of cardiac and renal disorders, the conjugate may be simplyadministered, generally by a parenteral route. In certain embodiments,administration of the conjugate is preceded by an administration of anIGFBP-derived internalization peptide in the absence of the therapeuticmolecule. Such pretreatment may be carried out for any convenient periodof time, such as about 2 days to about 4 weeks, or 4 days to threeweeks, or one week to 18 days prior to administration of the therapeuticmolecule/IGFBP-derived internalization peptide conjugate. For example,immediately following myocardial infarction molecules designed toprevent death of cardiomyocytes, such as appropriate pro-survival genes(e.g., anti-sense oligonucleotides which inhibit the expression ofbax-alpha or caspase-3, or polynucleotides which direct the expressionof bcl-2) may be systemically administered. In other embodiments, suchconjugates may be administered to ameliorate the effects of renalinsults, such as ischemia, hepatic insults, such as drug overdose (e.g.,acetominophen or acetominophen in combination with alcohol) or exposureto hepatotoxic chemicals (e.g., by ingestion of toxic mushrooms such asAmanita phalloides). Conjugates of the invention may also be used todeliver therapeutic molecules to skeletal muscle and cells of the immunesystem, exploiting the localization of MBD peptides to muscle andspleen, respectively.

The invention also provides methods of treating disorders which areassociated with altered iron metabolism, such as neoplasia, Parkinson'sdisease, and atherosclerosis. An IGFBP-derived internalization peptide(e.g., an MBD peptide) conjugated with a therapeutic molecule isadministered to a subject having a disorder associated with altered ironmetabolism, resulting in amelioration of at least one symptom of thedisorder. In certain embodiments, the IGFBP-derived internalizationpeptide further comprises a caveolin consensus binding sequence. Forexample, the increased iron uptake in neoplastic cells, Parkinson'sdisease, and atherosclerosis may be exploited to deliver therapeuticmolecules to the affected cells in these disorders in the form of aconjugate with an IGFBP-derived internalization peptide (e.g. a MBDpeptide).

Molecules comprising an IGF-binding protein derivative peptide or smallmolecule are preferably administered via oral or parenteraladministration, including but not limited to intravenous (IV),intraperitoneal (IP), intramuscular (IM), subcutaneous (SC), intradermal(ID), transdermal, inhaled, and intranasal routes. IV, IP, IM, and IDadministration may be by bolus or infusion administration. For SCadministration, administration may be by bolus, infusion, or byimplantable-device, such as an implantable minipump (e.g., osmotic ormechanical minipump) or slow release implant. The IGF-binding proteinderivative peptide or small molecule may also be delivered in a slowrelease formulation adapted for IV, IP, IM, ID or SC administration.Inhaled IGF-binding protein derivative peptide or small molecule ispreferably delivered in discrete doses (e.g., via a metered dose inhaleradapted for protein delivery). Administration of a molecule comprisingan IGF-binding protein derivative peptide or small molecule via thetransdermal route may be continuous or pulsatile. Administration ofderivative peptides or small molecules may also occur orally.

For parenteral administration, compositions comprising an IGF-bindingprotein derivative peptide or small molecule may be in dry powder,semi-solid or liquid formulations. For parenteral administration byroutes other than inhalation, the composition comprising an IGF-bindingprotein derivative peptide or small molecule is preferably administeredin a liquid formulation. Compositions comprising an IGF-binding proteinderivative peptide or small molecule formulations may contain additionalcomponents such as salts, buffers, bulking agents, osmolytes,antioxidants, detergents, surfactants, and other pharmaceuticalexcipients as are known in the art.

A composition comprising an IGF-binding protein derivative peptide orsmall molecule is administered to subjects at a dose of about 0.001 toabout 40 mg/kg/day, more preferably about 0.01 to about 10 mg/kg/day,more preferably 0.05 to about 4 mg/kg/day, even more preferably about0.1 to about 1 mg/kg/day.

As an alternative to administration of a composition comprising an IGFBPderivative peptide or small molecule, a nucleic acid construct encodingthe a composition comprising an IGFBP derivative peptide or smallmolecule may be administered. The construct contains a polynucleotidesequence encoding the composition comprising the IGFBP derivativepeptide, and normally contains sequences operably linked to the IGFBPderivative peptide sequence which result in expression and translationof the composition comprising the IGFBP derivative peptide sequence inthe cells (e.g., a promoter/enhancer, translation initiation site,polyadenylation signal, etc.), although constructs which are designed tointegrate into the cell chromosome are also contemplated (e.g., wherethe construct contains sequence which facilitates integration into thehost chromosome, such as sequences homologous to the recipient cells'chromosome flanking the IGFBP derivative peptide sequence).

Methods of gene transfer are well known in the art, and include in vitromethods (e.g., transformation of cultured cells, preferably autologouscells, which are reintroduced into the subject), ex vivo methods (e.g.,transformation of cells which have not been cultured in vivo, preferablyautologous cells, with are reintroduced into the subject), and in vivomethods (e.g., transformation of cells in situ by administration of anucleic acid construct to the subject). Methods for accomplishing suchgene transfer are well known in the art, and include standardtransformation methods including calcium phosphate transformation,ballistic transformation, electroporation, lipid-mediatedtransformation, naked DNA transfer, and viral-mediated transfer (e.g.,adenovirus and adeno-associated virus vectors).

The composition comprising an IGF-binding protein derivative peptide orsmall molecule is administered to the subject together with one or moreof the following co-administered agents: a chemotherapeutic agent; anantibody; physical stress, such as radiation; a treatment regimen, suchas a nutritional regimen; or a ligand of a receptor present on thetarget cells, such as retinoid receptors and thyroid receptors. Theadministration of the two agents may be simultaneous, overlapping, orseparated in time, as long as the subject experiences exposure to bothagents at the same time. Where the two agents are formulated for thesame route and schedule of administration, the administration ispreferably simultaneous or nearly simultaneous (e.g., concurrent orserial injections). However, in some embodiments, the routes andschedules of administration for the two agents will be different, makingsimultaneous administration inconvenient. A subject will be consideredto have been administered both agents if the subject experiencessimultaneous systemic exposure to both compounds, regardless of when orhow the compounds were administered.

In methods requiring the administration of co-administered agent withthe composition comprising an IGF-binding protein derivative peptide orsmall molecule, the dose of the co-administered agent is normallytitrated for the individual subject, as is known in the art for thatagent. Co-administered agents may be produced in any formulation knownto the art, including parenteral and oral dosage forms. Oralformulations are preferred, but parenteral formulations are alsoacceptable, and may be more convenient in an in-patient setting.Formulations for parenteral administration are generally formulated asliquids, but may also be in gel or solid depot form. Formulations fororal administration are generally in tablet or capsule form, althoughsyrups and liquids are also acceptable. Formulations of co-administeredagents generally include excipients, such as salts, buffers, bulkingagents, detergents, binding agents, surfactants, stabilizers,preservatives, anti-oxidants, lubricants, coating agents, and otherpharmaceutically acceptable excipients as are known in the art.

The dosage and mode of administration of the co-administered agentshould be adjusted according to the identity, formulation, route ofadministration and other relevant characteristics pertaining to theco-administered agent, as is known in the art.

Inducers and antagonists would be administered in a similar way. As anexample: Where the antagonist is propylthiouracil, the dose ofpropylthiouracil may be from 1 to 400 mg/day. A subject is normallyinitiated with a dose, of 50 to 400 mg/day, typically divided into threeequal doses, and maintained at 50 to 100 mg/day divided into two orthree equal doses. For methimazole and carbimazole, the dose may be from0.1 to 50 mg/day. Typically, a subject is initiated with 5 to 50 mg/day,and maintained on 1 to 5 mg/day.

As will be understood by those of skill in the art, the symptoms ofdisease alleviated by the instant methods, as well as the methods usedto measure the symptom(s) will vary, depending on the particular diseaseand the individual patient.

Patients treated in accordance with the methods of the instant inventionmay experience alleviation of any of the symptoms of their disease. Forexample, cancer patients treated with a MBD peptide and aco-administered agent may experience tumor stabilization (e.g., may failto progress), tumor shrinkage, or tumor elimination. Treatment with MBDpeptide and a co-administered agent may also result in reduced incidenceof metastasis or reduced numbers of metastatic tumors. Cardiovasculardisease patients treated with MBD peptide may experience reduction orelimination of any of their symptoms, including vascular stenosis andangina, and/or a reduction in number, size or formation ofatherosclerotic plaques. Patients having autoimmune disease who aretreated with MBD peptides may experience reduction, elimination, orstabilization of any of their symptoms, such as renal flares, fatigue,weight loss, arthralgia, butterfly rash, anemia (SLE), or weakness,paresthesia, number or size of sclerotic brain lesions (MS), or pain,stiffness, swelling or improvements or stabilization of range of motion(arthritis, particularly rheumatoid arthritis), or abdominal/epigastricpain, cramping, diarhea (inflammatory bowel disease including Crohn'sdisease), or fasting serum glucose levels or insulin requirement(diabetes), or wheezing, coughing, peak expiratory flow rate or need for‘rescue’ medications such as beta agonists (asthma), or allergicrhinitis (allergy). Retinopathy patients treated with MBD peptides mayexperience improvement or stabilization in visual acuity. Patientshaving bone resorptive diseases such as osteoporosis may experience animprovement, stabilization, or decrease in rate of loss of bone mass,which may manifest itself as a reduced risk or rate of bone fracture.

Kits

The invention provides kits comprising IGFBP derived peptides or smallmolecules. The kits comprise at least one package comprising acomposition comprising an IGFBP derived peptide or small molecule.Optionally, the kits may also include a set of instructions for use ofthe composition.

The compositions included within the kits may be an IGFBP derivedpeptide or small molecule, or a fusion/conjugate comprising an IGFBPderived peptide or small molecule. In certain embodiments, the kit mayalso contain at least one package of a co-administered agent, such as acytotoxic chemotherapy drug (e.g., paclitaxel or doxorubicin). Thecontainers of the composition comprising the IGFBP derived peptide orsmall molecule (and optional co-administered agent) may be unit doses,bulk packages (e.g., multi-dose packages) or sub-unit doses.

In embodiments comprising instructions, the instructions generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended use of the included composition(s)(e.g., for treatment of cancer, hyperproliferative disorders, orarterial restenosis). Instructions supplied in the kits of the inventionare typically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

The patents, patent applications, and publications cited throughout thedisclosure are incorporated herein by reference in their entirety.

EXAMPLES Example 1 Treatment of Nutritionally Stressed HEK293 KidneyCells with IGFBP-3

Human embryonal kidney 293 (HEK293) cells were grown in Dulbecco'sModified Eagle Medium (D-MEM) supplemented with fetal calf serum at 2%,4%, 6%, or 8%. When the cells reached 80-85% confluency (cell titerapproximately 2.1×10⁶ cells per plate), 5 μg of IGFBP-3 or buffercontrol was added to each plate. The cells were incubated at 37° C.overnight. The next day the medium was removed and the cells were rinsedwith trypsin-EDTA (0.25% trypsin, 1 mM EDTA) plus 1× phosphate bufferedsaline. The cells were centrifuged and the supematant was removed.ApoAlert caspase-3 assay kit from Clontech Inc (Palo Alto, Calif.) wasused to measure apoptosis. The cells were resuspended in 50 μl ofchilled cell lysis buffer and incubated on ice for 10 minutes. Theresulting cell lysates were centrifuged at 14000 rpm in a Beckmanmicrocentrifuge for 3 minutes at 4 C. The supernatant was transferred tonew tubes and 50 ul of 2× reaction buffer/DTT plus 5 μl of 1 mMcaspase-3 substrate was added to each tube. After incubating at 37 C for1 hour in a water bath, the samples were read at 405 nm in a microplatereader. The results of this experiment are shown in FIG. 3.

Example 2 Identification of Pro-Apoptotic Peptide Sequences

As shown in FIG. 4A and disclosed in co-owned U.S. patent applicationSer. No. 09/956,508, IGFBP-3 has pro-apoptotic activity. Peptidesderived from IGFBP-3 were tested for pro-apoptotic activity essentiallyas described in Example 1.

The peptides tested are described in Table 1 (peptides marked with anasterisk include a hexahistidine tag). TABLE 1 Peptide reference Control(no peptide) A rh (N109D)-IGFBP-3 B glycosylated CHO-derived rhIGFBP-3 CrhIGFBP-3/rhIGF-I complex D rhIGFBP-3/rh(Y60L)-IGF-I complex E*(KKGHAKDSQRYKVDYESQS)-gfp F *(KKGFYKKKQCRPSKGRKRGFCW)-gfp GDKKGFYKKKQCRPSKGRKRGFCW H QCRPSKGRKRGFCW I

Data from this experiment, summarized in FIG. 4B, demonstrated, for thefirst time, that it was possible to generate IGFBP-3-derived peptidesexhibiting greater pro-apoptotic activity (on a weight basis) thanintact IGFBP-3 itself. Comparing the pro-apoptotic activity of peptidesH and I, it is remarkable that peptide I exhibited 3-4× higherpro-apoptotic activity on a weight basis than intact IGFBP-3 (B, C) or alonger peptide (G, H). That is, the presence of 9 additional amino acidsin peptide H compared to peptide I resulted in a dramatic lowering ofpro-apoptotic activity.

Example 3 Metal-Binding Properties of IGFBP-3 and Derivative Peptides

As shown in FIG. 5, intact IGFBP-3 binds immobilized nickel and zinc andcan be eluted from the resin with 60 mM imidazole. This previouslyunknown property of IGFBP-3 is intriguing and has a number of practicalimplications among which the ability to capture products usingimmobilized metal affinity (IMAC) resins leads to a number of potentialapplications in therapeutic, high-throughput discovery, and otherresearch arenas.

FIG. 5 shows SDS-PAGE results from IMAC purification of IGFBP-3 usingNi⁺⁺ (panel a) and Zn⁺⁺ (panel b) IMAC. IGFBP-3 bound both IMAC resinseffectively.

We further tested the ability of IGFBP-3-derived sub-domains andpeptides to bind metal. As shown in Example 5, below, a defined fragmentof IGFBP-3 generated in vivo can be captured on IMAC. Two short peptideswere passed through a Ni-His-Bind column: Peptide 1:(H2N)...DKKGFYKKKQCRPSKGRKRGFCW...(COOH); Peptide 2:(H2N)...QCRPSKGRKRGFCW...(COOH)

Both peptides bound specifically to the column. Peptide 1 eluted with 60mM imidazole, whereas higher concentrations of imidazole (1M) werenecessary to elute Peptide 2. It therefore appears that Peptide 2 bindsmetal more tightly than does Peptide 1.

Example 4 Treatment of LAPC-4 Prostate Humor Cells with IGFBP-3 andCo-Administered Agents

A study was performed to analyze the effects of IGFBP-3 in combinationwith TAXOL® on the growth and death of prostate cancer cells utilizingthe LAPC-4 xenograft model. One million cells (in 100 μl) were injectedSQ into SCID mice. After 4 weeks palpable tumors were observed. 4 groupswere treated (6 mice per group): 1) saline control; 2) IGFBP-3 (4mg/kg/day intra-peritoneally); 3) TAXOL® (2 mg/kg/day intra-peritoneallyon days 5 through 8); 4) TAXOL® and IGFBP-3 combination. Tumors wereanalyzed for size by palpation weekly and serum collected. Animals weresacrificed at day 21 and tumor weight assessed. The results of thisexperiment demonstrated a trend for reduced tumor size (40%) withcombination therapy. This biological action is believed to result fromthe pro-apoptotic activity of IGFBP-3.

Example 5 Generation of Defined Sub-Domains of IGFBP-3 by Engineering 3CProtease Target Sites into the Primary Sequence of the Protein

Defined IGFBP-3 sub-domains were generated from constructs expressed assoluble fusion proteins in an E. coli expression system. The generalstructures of the fusions are:

-   -   IVS-1: DsbA(mut) . . . [3C] . . . domain 1 . . . [3C] . . .        domain 2/3    -   IVS-2: DsbA(mut) . . . [3C] . . . domain 1/2 . . . [3C] . . .        domain 3

where [3C] is the peptide sequence recognized by HRV 3C proteinase. Thegeneral strategy for generating defined domains is shown in FIG. 5.Yields are comparable to wild type, and a substantial fraction isbelieved to be correctly folded, based on the demonstrated ability ofthe protein to bind IGF-I. After cleavage, the sub-domains of IGFBP-3generated from the IVS-i construct (domains 1, 2/3) are captured onhydrophobic interaction resins such as Phenyl-SEPHAROSES or (lessdesirably) on cation exchange resins such as SP-SEPHAROSES. Otherresins, such as immobilized heparin can also be used. Efficienton-column cleavage of IVS-1 fusion with 3C proteinase has beendemonstrated using 1:10 (protease to substrate) ratios at 4 degreesCelsius or room temperature. Complete cleavage has been seen in lessthan 20 minutes. In the past, amino acid sequencing of cleavage productshas shown that the enzyme cleaves in an unusually clean manner (<5%“ragged” ends). Further purification to near homogeneity can be achievedon nickel- or zinc-affinity chromatography. Apparently, metal-bindingdoes not require the amino-terminal ˜100 amino acids of the protein,which are believed to constitute the primary domain for IGF-I binding inthe IGFBP-3 molecule.

Example 6 Identification of Cell Internalization Peptide

Three peptide extensions were each cloned in frame with the gene forgreen fluorescent protein (gfp; Clontech) and expressed in E. coli(JM109). Each construct further contains a 6H tag. Products werecaptured on His-Bind Resin (Novagen) and eluted with 60 mM imidazole0.5M NaCl, then further purified on a HIC (Phenyl-SEPHAROSE® HighPerformance Resin, Amershamn) resin and eluted with 50 mM PhosphateBuffered Saline. The purified peptide-gfps were tested for cellinternalization on HEK293, a human embryonal kidney cell line. HEK293cells were cultured in Dulbecco's Modified Eagle Medium until they were80-85% confluent (˜2.1×10{circumflex over ( )}6 cells per plate). Freshmedium containing each peptide (11 μg per plate) was added to theplates. The cells were incubated at 37° C. for approximately thirtyminutes. The medium was removed, the cells were trypsinized and washedwith 1× Phosphate Buffered Saline twice. Cells were held under alongwave UV lamp to determine fluorescence. Photographs of the samplesare shown in FIG. 7. Sample “d” was the only sample that exhibitedstrong fluorescence. It thus appears that the peptideKKGFYKKKQCRPSKGRKRGFCW contains all the sequence information necessaryto direct the internalization of a large, unrelated protein into HEK293cells. Combined with the known nuclear translocation properties believedto reside in part of this same sequence, this peptide may be useful as acell uptake and nuclear transporter for a variety of molecules such asproteins, nucleic acids and small chemical moieties. Applications inconventional and gene therapy, cell imaging, research, andhigh-throughput screening are envisaged.

Example 7 Metal-Binding Properties of IGFBP-3

Binding of IGFBP-3 to immobilized metal affinity chromatography (IMAC)resin charged with various metals was measured. Approximately 1 mg ofIGFBP-3 was loaded onto each column (NTA resin, Sigma Chemical Co., St.Louis, Mo.). The “percentage bound” was calculated by measuring OD280 ofload, flow-through, wash and eluate at 60 mM imidazole. Typicalrecoveries were 85-95%. Results are summarized in Table 2. TABLE 2 Metallog stability K % IGFBP-3 bound Ni⁺⁺ 11.5 97.6 Co⁺⁺ 10.8 95.1 Zn⁺⁺ 10.459.5 Fe³⁺ 15.9 55.1 Fe²⁺ NA 46.6 Mg⁺⁺ 5.5 38.8 Mn⁺⁺ 7.4 20.1 Ca⁺⁺ 6.50.0 none 0.0

Example 8 Co-Apoptotic Activity of IGFBP-3 and Paclitaxel

Co-aptotic activity of IGFBP-3 in combination with paclitaxel wasmeasured in the HEK293 assay described in Example 1. HEK293 cells weregrown in DMEM plus 8% FCS, then incubated in 0.3 ng/ml of paclitaxel(TAXOL®), 50 ng/ml of IGFBP-3, or a combination of the two. Somecultures were pretreated for 30 minutes with 200 ng/ml ofanti-beta-1-integrin antibody (Pharmingen). Caspase-3 was assayed usingthe ApoAlert caspase-3 kit from Clontech Inc.

Results of the experiment shown in FIG. 8. The results shown in FIG. 8Ademonstrate the strong co-apoptotic synergy of paclitaxel and IGFBP-3 onHEK293 cells. Pretreatment with anti-beta-1-integrin antibody greatlyinhibits the co-apoptotic activity of IGFBP-3 in this assay, as shown inFIG. 8B. However, IGFBP-3 failed to demonstrate any co-apoptoticactivity in a similar experiment performed using MDA-MB-231 cells andcisplatin (FIG. 8C).

Example 9 Rapid Cellular Uptake of GFP Directed by MBD2 Peptide

Polynucleotides encoding the peptides KKGHAKDSQRYKVDYESQS (irrelevantpeptide GFP31), KKGFYKKKQCRPSKGRKRGFCW (long peptide GFP32), KKGFYKKK(upstream peptide GFP34), and QCRPSKGRKRGFCW (downstream peptide GFP35which includes MBD2) were cloned as in-frame fusions to the 5′ end ofthe GFP coding sequence in the pGFPuv vector (Clontech Inc., Palo Alto,Calif.). Expressed proteins were purified via metal-affinity andhydrophobic interaction chromatography.

Each protein was added to 80% confluent HEK293 cells at 0.5 μg/ml. FIG.9A shows, from top to bottom, a Coomassie stained gel of the purifiedproteins, GFP fluorescence of treated cells 1 hr post-addition and aWestern blot of extracts from the same cells (duplicate experiments).Westerns were probed with anti-GFP antibody. FIG. 9B further showsfluorescence of GFP32-treated cells indicating that uptake of GFP32 intothese cells can be selectively inhibited by pretreating the cells with200 ng/ml of anti-integrin but not anti-fibronectin antibodies.

Example 10 Co-Apoptotic Activity of MBD Peptides and Paclitaxel

Peptides (50 ng/ml) were added to 80% confluent HEK 293 cells grown inDMEM supplemented with 0.3 ng/ml paclitaxel concurrent with peptide.Caspase-3 activity was measured in cell extracts 8 hours post-addition.

Results are summarized in Table 3. Apoptotic activity is expressed inarbitrary caspase-3 units normalized to MBD2 on a molar basis (MBD2activity was defined as 100 units (avg. of 3 experiments); “nd”indicates “not done”; “ps” indicates phosphoserine. Metal-binding isexpressed as a percentage of loaded peptide bound to a Ni—NTA resin;approximately 1-1.5 mg of peptide were loaded on the column. Cell uptakewas determined using genetic fusions to GFP as described in Example 9.TABLE 3 Apoptotic Metal- Cell Peptide Sequence Activity* binding UptakeBP3 full-length, 264 aa, IGFBP-3 585 93 +++ MBD1  DKKGFYKKKQCRPSKGRKRGFCW 10-15 22 +++ MBD9   KKGFYKKKQCRPSKGRKRGFCWNGRK 149 45 nd MBD5      GFYKKKQCRPSKGRKRGFCW10-15 95 nd MBD2            QCRPSKGRKRGFCW 100 66 ++ MBD2*PS           QCRP(ps)KGRKRGFCW nd nd nd MBD3            QCRPSEGGKRGFCW <5100 nd BP2nls            QCKMSLNGQRGECW <5 nd nd BP5nls           QCKPSRGRKRGICW <5 28 nd MBD12             CRPSKGRKRGFCW 10-40nd nd MBD13            QCRPSKGRKRGFC 10-40 nd nd MBD4            CRPSKGRKRGFC 10-15 92 nd MBD7            QCRPSKGRKRGFCWNGR89 11 nd MBD10 KKEKDIMKKTIQCRPSKGRKRGFCWNGR 127 nd nd

Example 11 Antigenic Profiling of MBD Peptides

The antigenic profiles of MBD peptides were assayed by ELISA. MBDpeptides or IGFBP-3 were added to wells of a 96-well Ni—NTA plate(Qiagen Inc., Carlsbad, Calif.) for 15 minutes washed twice in PBS Tweenbuffer, then blocked for 4 hours with 3% BSA in the same buffer.Detection was done colorimetrically (recorded in absorbance units) usinga second antibody conjugated to horse-radish peroxidase.

As shown in FIG. 10, MBD peptides are antigenically distinct from fulllength IGFBP-3. None of the MBD peptides tested reacted with polyclonalanti-IGFBP-3 antibody, as shown in panel (a). Panel (b) shows theabsorbance units obtained using a polyclonal antibody raised to MBD5peptide, which bound to the MBD peptides, but not IGFBP-3.

Example 12 MBD Co-Apoptotic Activity is Resistant to Inhibition byPlasma Proteins

Co-apoptotic activity of MBD peptides or IGFBP-3 with paclitaxel wastested in the presence or absence of two plasma proteins; fibronectinand fibrinogen. MBD peptides or IGFBP-3, (50 ng/ml), 0.3 ng/mlpaclitaxel, and plasma proteins (200 ng/ml) were added to 80% confluentHEK293 cells grown in DMEM essentially as described in Example 8.Caspase-3 was measured after an eight hour incubation.

Apoptotic activity was calculated in arbitrary caspase-3 unitsnormalized to MBD2 (MBD2 activity was defined as 100 units) Results aresummarized in Table 4 (average of 3 experiments). Fibronectin andfibrinogen inhibit IGFBP-3 co-apoptotic activity is essentiallyeliminated by both fibronectin and fibrinogen, while MBD2 co-apoptoticactivity is either essentially unaffected (fibronectin) or onlymoderately reduced (fibrinogen). TABLE 4 Plasma Protein MBD2 IGFBP-3none 100.0 100.0 Fibronectin 98.3 3.5 Fibrinogen 67.5 1.1

Example 13 Treatment with IGFBP-3 Plus Doxorubicin or 5-FluorouracilReduces Tumor Size

Fragments of MA-16C mammary adenocarcinoma tumors were implantedsubcutaneously into female C3H mice (8-11 animals per group). Theanimals were treated with vehicle alone, IGFBP-3 (4 mg/kg/day for 21days by subcutaneous injection), doxorubicin (2 mg/kg on days 1 and 8 byintravenous injection), or IGFBP-3 plus doxorubicin. A separate studywas performed where the animals were treated with vehicle alone,5-fluorouracil (10 mg/kg on days 1-5 by intraperitoneal injection), orIGFBP-3 plus 5-fluorouracil. Tumor size was measured at day 18, and themice were sacrificed at day 21, and the implanted tumors were removedfor determination of caspase-3 activity (a marker of apoptosis-onlyperformed for DOX groups).

Treatment with IGFBP-3 combined with either doxorubicin (DOX) or5-fluorouracil (5FU) significantly reduced tumor size as compared totreatment with vehicle or IGFBP-3 alone (p<0.01). Additionally, tumorshrinkage by IGFBP-3 plus DOX was significant when compared to the DOXalone group (p<0.02). Results for the DOX groups are summarized in Table5 and results for the 5FU groups are summarized in Table 6. TABLE 5Treatment Tumor Weight (mg) Caspase-3 Lung Metastases Vehicle 2878 ±1025 4.42 + 3.77 4/8 DOX 1966 + 677  7.88 + 6.27  0/12 IGFBP-3 2632 +554  3.79 + 3.25 1/7 IGFBP-3/DOX 1033 + 831  9.02 + 2.81 3/9

TABLE 6 Treatment Tumor Weight (mg) Lung Metastases Vehicle 3198 + 11304/8 5FU 2595 + 1767  4/10 IGFBP-3 3147 + 952  1/7 IGFBP-3/5FU 1779 +723   1/10

Example 14 Stimulation of Apoptosis by MBD Peptides is IGF-Independent

HEK 293 cells were cultured under stressed conditions (low serum or inthe presence of paclitaxel) with and without IGFBP-3 (50 ng/ml) or MBD2(50 ng/ml). Apoptosis was assayed using the caspase-3 assay as describedin Example 1. As shown in FIGS. 11A and 11B, the pro-apoptoticactivities of IGFBP-3 and MBD2 are nearly identical.

IGF-dependence was investigated using HEK 293 cells treated with 0.3ng/ml of paclitaxel in the presence of IGFBP-3 or MBD2, both alone or incombination with Y60L-IGF-I (an IGF-I mutant that does not bind to theIGF receptor). As shown in FIG. 11C, the pro-apoptotic activity ofIGFBP-3 and MDB2 are independent of the presence of IGF.

Example 15 The Pro-Apoptotic and Cell Internalization Activities of MBDPeptides are Integrin Dependent

HEK 293 cells were cultured as described in Example 10, includingpaclitaxel. Cells were incubated with MDB2 either alone or incombination with an anti-adhesion protein antibody. Antibodies wereagainst integrin associated protein (LAP), fibronectin (Fn), transferrinreceptor (TfnR), alpha 5 integrin, alpha 6 integrin, alpha v integrin,beta 1 integrin, and beta 5 integrin. Caspase-3 activity was assayed asdescribed in Example 1.

Results are summarized in FIG. 12A, the pro-apoptotic activity of MBD2is inhibited by antibodies against integrins and integrin-associatedproteins.

Example 16 The Pro-Apoptotic Activity of MBD Peptides isSequence-Specific

HEK 293 cells were cultured and assayed as described in Example 10. MBD2and 6 MBD2 (MBD2(ps) has a phosphoserine at position 5) variants wereassayed for co-apoptotic activity. Results are summarized in FIG. 13,which shows that the co-apoptotic activity of MDB peptides is highlysequence specific to the core sequence.

Example 17 Expression of Bax-Alpha is Stimulated by IGFBP-3 and MBDPeptides

HEK 293 cells were cultured as described in Example 1, with the additionpaclitaxel (0.3 ng/ml) and either IGFBP-3 or MBD2 at 50 ng/ml. Bax-alphaand bcl-2 mRNA and caspase 3 activity were assayed. As summarized inFIG. 14A, IGFBP-3 stimulates bax-alpha expression. Bax-alpha expressionis correlated with caspase-3 activity, as shown in FIG. 14B.

Example 18 MBD Co-Apoptotic Activity is Dependent on PI3K/ILK SignalTransduction

HEK 293 cells were transfected with pUSEamp, or pUSEamp carrying a Aktdominant negative (AktDN), PTEN, ILK dominant negative (ILKdn) or rafdominant negative (rafDN) insert (Upstate Biotechnologies). The cellswere cultured as described in Example 10 (including paclitaxel), with orwithout MBD2. As summarized in FIG. 15, MBD2 pro-apoptotic activity isinhibited in cells in which the PI3K/ILK kinase signal transductionpathway is blocked, but not in cells where the MAPK signal transductionpathway is blocked.

Example 18 MBD Peptides Sensitize Cancer Cells to Low Doses of CytotoxicChemotherapy Agents

MA16C cells were cultured in the presence of MDB2 and a low dose ofdoxorubicin (75 ng/ml). Apoptosis was assayed using the caspase-3 assayas described in Example 1. As summarized in FIG. 16A, MDB2 sensitizesMA16C mammary adenocarcinoma cells to low doses of doxorubicin.

MDA-MB-231 cells were cultured in the presence of low dose paclitaxel(100 ng/ml) with and without MDB2 (50 ng/ml). Apoptosis was assayed bymeasuring NMP levels. As summarized in FIG. 16B, MDB2 sensitizesMDA-MB-231 cells to low doses of paclitaxel.

Example 19 Ferrous Iron Unmasks MBD Peptide Epitopes

One milligram of pure IGFBP-3 was incubated at room temperature in (a)PBS (b) 100 μM ferrous chloride in PBS, (c) 100 μM ferric chloride inPBS, or (d) 100 μM zinc chloride in PBS, either in the presence orabsence of 200 ng IGF-I. Aliquots were withdrawn at 0, 20, 40 and 60minutes, then assayed by ELISA using an anti-MBD antibody.

Assay results, summarized in FIG. 17, indicate that incubation ofIGFBP-3 with ferrous iron (Fe⁺⁺), but not ferric iron (Fe⁺⁺⁺) or zinc(Zn⁺⁺) resulted in the unmasking of MBD peptide epitopes that are notnormally detectable on full length IGFBP-3.

Example 20 Differential Localization of MBD Peptides

1 mg/ml MBD-GFP32 protein was pre-incubated in (a) PBS (b) 100 μMferrous chloride in PBS, (c) 100 μM ferric chloride in PBS, or (d) 100μM zinc chloride in PBS for 60 minutes. 20 minutes after addition of 500ng/ml MBD-GFP32 to the cell culture medium, nuclear extracts of HEK293cells were prepared using a Nuclear Extract Kit from Sigma according tothe manufacturer's instructions. Extracts were assayed by anti-GFPELISA.

Assay results indicate that preincubation of MBD-GFP32 with ferrousiron, but not ferric iron or zinc, inhibits nuclear localization of theprotein. Results are summarized in FIG. 18.

Example 21 Nuclear Translocation of MBD Peptides Involves Caveolin- andClathrin-Mediated Pathways

HEK293 cells were pre-incubated with inhibitors nystatin (inhibitor ofcaveolin pathway) or chlorpromazine (inhibitor of clathrin pathway) forone hour at 10 μg/ml. Twenty minutes after addition of 500 ng/mlMBD-GFP32 to the cell culture medium, nuclear extracts of the cells wereprepared as described in Example 20. Extracts were assayed by anti-GFPELISA.

As summarized in FIG. 19, both nystatin and chlorpromazine inhibitnuclear translocation of MBD peptides, suggesting that nucleartranslocation of MBD peptides is mediated by both the caveolin andclathrin mediated endocytosis pathways. Results shown are averages ofthree plates per data point.

In an additional set of experiments, HEK293 cells were pre-incubatedwith no inhibitor (control), nystatin or chlorpromazine (10 μg/ml) for60 minutes, then cooled to 4° Celsius. Cells were gently removed fromthe plate in the absence of trypsin and incubated in 500 ng/mlMBD-GFP32. Cross-linker B3 was added for 10 minute pulses at varioustimes after the addition of MBD-GFP32 to the cells. Cross-linkingreactions were terminated with 100 mM Tris buffer pH 8.0. Cell extractswere captured on Nickel-NTA-coated 96-well plates, then assayed by ELISAusing antibodies to GFP (for standardization of samples), transferrinreceptor, or alpha 5 integrin.

Assay results, summarized in FIGS. 20 and 21, show that both nystatin(an inhibitor of the caveolin-mediated endocytosis pathway) andchlorpromazine (an inhibitor of the clathrin-mediated endocytosispathway) inhibit association between MBD peptides and cell surfacemarkers. Results shown are averages of triplicates.

Example 22 Differential Localization of MBD Peptides

Female C3H mice were pretreated for 18 days with either subcutaneousinjections of MBD7 peptide (2 mg/kg/day) or saline. A single intravenousbolus of MBD-GFP32 or MBD-GFP37 (MBD37 peptide,KKGFYKKKQCRPSKGRKRGFCWNGR linked to GFP) protein was administered at 2mg/kg, followed by sacrifice and tissue harvest two hours later.

Tissue extracts were prepared and analyzed by anti-GFP ELISA. Assummarized in FIGS. 22 and 23, the distribution of MBD peptides ishighly unusual, and is altered by pretreatment with MBD peptide.Preferential targeting to heart and kidney correlates well with dataobtained in iron-loading studies in which cardio and renal toxicitieshave been observed

Example 23 MBD-Peptide-Mediated Nuclear Translocation of Macromoleculesis Greatly Enhanced by Presence of Caveolin-Binding Consensus Sequence

Biotinylated MBD peptides MBD9, MBD20 (QCRPSKGRKRGFCWAVDKYG: the MBDcore 14 mer is underlined, and the caveolin consensus binding sequenceis bolded) or MBD21 (KKGFYKKKQCRPSKGRKRGFCWAVDKYG: the MBD core 14 meris underlined, and the caveolin consensus binding sequence is bolded)were each mixed with streptavidin-horseradish peroxidase conjugate (3-6×molar excess of MBD peptide) and added to HEK293 cells. After 20minutes, nuclear and cytoplasmic extracts were prepared as described inExample 20 and assayed for peroxidase activity.

Assay results, summarized in FIG. 24, show that efficient nuclearmobilization of this large (approx. 90 Kd) conjugate proceeded far moreefficiently when MBD20 or MBD21 was used, as opposed to MBD9. There isno detectable cytoplasmic peroxidase activity. The notable differencebetween the sequences of these peptides is the presence of an additional6 amino acids on the C-terminal side of the core MBD 14-mer sequence(present in the canonical MBD2 peptide) in both MBD20 and MBD21 but notMBD9. The presence of this sequence completes a motif known to bepresent in proteins that bind caveolin (#x#xxxx#, where ‘#’ is anaromatic amino acid). The-use of the MBD20 sequence to mobilize therapid uptake of a large molecule from outside living cells directly intothe nucleus is potentially very significant, particularly inapplications involving nucleic acids (such as gene therapy) as well asmodulators of nucleic acid activity.

The present invention has been detailed both by direct description andby example. Equivalents and modifications of the present invention willbe apparent to those skilled in the art, and are encompassed within thescope of the invention.

1. A method for alleviating the symptoms of a disease selected from thegroup consisting of autoimmune disease, cardiovascular indications,arthritis, asthma and allergy, reproductive indications, retinalproliferative disease, bone disease, inflammatory disease, inflammatorybowel disease, and fibrotic disease, comprising administering aneffective amount of an IGF-binding protein-derived peptide or smallmolecule to an individual suffering from said disease.
 2. The method ofclaim 1, wherein said IGF-binding protein-derived peptide or smallmolecule is administered at about 0.001 to about 40 milligrams perkilogram total body weight per day (mg/kg/day).
 3. The method of claim1, wherein said IGF-binding protein-derived peptide or small molecule isa MBD peptide.
 4. The method of claim 1, wherein said MBD peptidecomprises the sequence DKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO:1).
 5. Themethod of claim 1, wherein said MBD peptide comprises the sequenceGFYKKKQCRPSKGRKRGFCW (SEQ ID NO:2).
 6. The method of claim 1, whereinsaid MBD peptide comprises the sequence QCRPSKGRKRGFCW (SEQ ID NO:3). 7.The method of claim 1, wherein said MBD peptide comprises the sequenceQCRPSKGRKRGFC (SEQ ID NO:8).
 8. The method of claim 1, wherein said MBDpeptide comprises the sequence CRPSKGRKRGFCW (SEQ ID NO:9).
 9. Themethod of claim 1, wherein said MBD peptide comprises the sequenceCRPSKGRKRGFC (SEQ ID NO:4).
 10. The method of claim 1, wherein said MBDpeptide further comprises a caveolin consensus binding site.
 11. Themethod of claim 10, wherein said MBD peptide comprises the sequenceQCRPSKGRKRGFCWAVDKYG (SEQ ID NO:37).
 12. The method of claim 10, whereinsaid MBD peptide comprises the sequence KKGFYKKKQCRPSKGRKRGFCWAVDKYG(SEQ ID NO:38).